PARS imaging methods

ABSTRACT

A method for visualizing details in a sample including directing an excitation beam to an excitation location below a surface of the sample, to generate signals in the sample; directing an interrogation beam toward the excitation location of the sample; directing a signal enhancement beam to the sample, to raise a temperature of a portion of the sample by 5 Kelvin or less, compared to a temperature of the portion of the sample in absence of the signal enhancement beam; detecting a portion of the interrogation beam returning from the sample that is indicative of the generated signals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 37 CFR § 1.53(b) ofpending prior International Application No. PCT/IB2021/055380, filedJun. 17, 2021, which claims priority to U.S. Provisional PatentApplication No. 63/187,789, filed on May 12, 2021, U.S. patentapplication Ser. No. 17/010,500, filed on Sep. 2, 2020, and U.S.Provisional Patent Application No. 63/040,866, filed on Jun. 18, 2020,now expired, the entirety of each of which is incorporated herein byreference.

FIELD

This relates to the field of optical imaging and, in particular, to alaser-based method and system for non-contact imaging of samples such asindustrial materials or biological tissue in vivo, ex vivo, or in vitro.

BACKGROUND

Photoacoustic imaging techniques represent a powerful family ofmodalities which are capable of visualizing intrinsic endogenous opticalabsorption contrast within optically scattering media. In commonphotoacoustic architectures, nanosecond or picosecond laser pulses aredirected into a sample causing the generation of thermo-elastic inducedacoustic waves, which are then observed and reconstructed to form imagesof the optical absorption distribution. By carefully selecting thewavelength of the excitation source, absorption contrast of specificbiomolecules can be targeted. For example, 532 nm is a widely usedwavelength for targeting hemoglobin. These systems have proven to beefficacious in recovering clinically relevant biological structure fromwithin biological tissues. Some examples include vascular structuresfrom macro vessels to micro vessels, cellular structure, and lipid richplaques along with functional imaging including visualization of bloodoxygen saturation.

Photoacoustic imaging can be split into two main categories:Photoacoustic tomography (PAT) uses reconstruction-based imageformation, while photoacoustic microscopy (PAM) uses focused-based imageformation. In PAT, an unfocused optical beam excites the region ofinterest, and an array of transducers measures the generated ultrasoundwaves in multiple positions. PAM employs raster-scanning of optical andacoustic foci and forms images directly from recorded depth-resolvedsignals. PAM can be further classified into optical-resolution PAM(OR-PAM), where the optical focusing is much tighter than acousticfocusing, and acoustic-resolution PAM (AR-PAM), where the acousticfocusing is tighter. In all three embodiments, the acoustic signal istypically collected through an acoustically coupled transducer or otheracoustic- or acousto-optic resonator. In all cases the photoacousticsignals (which are commonly associated with generation of pressure andtemperature within the sample) can be recorded to form an imagerepresenting the optical absorption in the sample at the excitationwavelength in which the amplitude of the various recorded peaks impliesthe local optical absorption.

However, since conventional photoacoustic techniques require physicalcoupling to the sample, inappropriate for a wide variety of clinicalapplications such as ophthalmic imaging, intraoperative imaging,monitoring of wound healing, and many endoscopic procedures.

A recently reported photoacoustic technology known as photoacousticremote sensing (PARS) microscopy (US 2016/0113507, and US 2017/0215738)has solved many of these sensitivity issues through a novel detectionmechanism. Rather than detecting acoustic pressures at an outer surfaceonce they have propagated away from their source, PARS enables directdetection of excited photoacoustic regions. This is accomplished bymonitoring changes in material optical properties that coincide with thephotoacoustic excitation. These changes then encode various salientmaterial properties such as the optical absorption, physical targetdimensions, and constituent chromophores to name a few.

SUMMARY

According to an aspect, there is provided a thermally enhancedphotoacoustic remote sensing (TE-PARS) system for imaging a subsurfacestructure in the sample which provides absorption contrast within thesample.

The TE-PARS system comprises an excitation beam or collection of beamsconfigured to generate PARS signals in the sample at an excitationlocation or collection of locations; a signal enhancement beam orcollection of beams configured to modify the observation or generationof temperature and pressure signals, incident on the sample at theexcitation or interrogation location or collection of locations; aninterrogation beam or collection of beams incident on the sample at aninterrogation location or collection of locations; an optical system orcollection of systems that focuses or directs the excitation beam orcollection of beams at a first focal point or collection of focalpoints, the signal enhancement beams at a second focal point orcollection of focal points and the interrogation beams at a third focalpoint or collection of focal points, the first, second and third focalpoints or collection of focal points being below the surface of thesample; a portion of the interrogation and or signal enhancement beam orcollection of beams returning from the sample that is indicative of thegenerated PARS signals; an optical detector or collection of opticaldetectors to detect the returning portion or portions of theinterrogation and or signal enhancement beams; and a processing unit forinterpreting collected results.

Embodiments of TE-PARS may comprise several collections of PARS signalenhancement pathways, which may also function as detection pathways.

According to another aspect, there is provided a temperature sensingphotoacoustic remote sensing (TS-PARS) system for detecting thetemperature of a subsurface structure within a sample.

The TS-PARS system comprises an excitation beam or collection of beamsconfigured to generate PARS signals in the sample at an excitationlocation or collection of locations; an interrogation beam or collectionof beams incident on the sample at an interrogation location orcollection of locations; an optical system or collection of systems thatfocuses or directs the excitation beam or collection of beams at a firstfocal point or collection of focal points and the interrogation beams ata second focal point or collection of focal points, the first and secondfocal points or collection of focal points being below the surface ofthe sample; a portion of the interrogation beam or collection of beamsreturning from the sample that is indicative of the generated PARSsignals; an optical detector or collection of optical detectors todetect the returning portion or portions of the interrogation and orsignal enhancement beams; and a temperature processing unit forinterpreting collected results.

According to another aspect, there is provided a super-resolutionphotoacoustic remote sensing (SR-PARS) system for imaging a subsurfacestructure in the sample with resolution greater than that defined by theoptical diffraction limit by leveraging optical absorption contrastwithin the sample.

The SR-PARS system comprises an excitation beam or collection of beamsconfigured to generate PARS signals in the sample at an excitationlocation or collection of locations; an interrogation beam or collectionof beams incident on the sample at an interrogation location orcollection of locations; an optical system or collection of systems thatfocuses or directs the excitation beam or collection of beams at a firstfocal point or collection of focal points and the interrogation beams ata second focal point or collection of focal points, the first and secondfocal points or collection of focal points being below the surface ofthe sample; a portion of the interrogation beam or collection of beamsreturning from the sample that is indicative of the generated PARSsignals; an optical detector or collection of optical detectors todetect the returning portion or portions of the interrogation beams; anda super-resolution processing unit for interpreting collected results.

According to another aspect, there is provided a spectrally-enhancedphotoacoustic remote sensing (SE-PARS) system for imaging a subsurfacestructure in the sample which leverages chromatic effects and spatialfiltering methods to encode spatial information within the sample.

The SE-PARS system comprises an excitation beam or collection of beamsconfigured to generate PARS signals in the sample at an excitationlocation or collection of locations; an interrogation beam or collectionof beams incident on the sample at an interrogation location orcollection of locations; an optical system or collection of opticalsystems which disburses the interrogation beams based on theirwavelength or spatial positioning; an optical system or collection ofsystems that focuses or directs the excitation beam or collection ofbeams at a first focal point or collection of focal points and theinterrogation beams at a second focal point or collection of focalpoints, the first and second focal points or collection of focal pointsbeing below the surface of the sample; a portion of the interrogationbeam or collection of beams returning from the sample that is indicativeof the generated PARS signals; an optical system or collection ofoptical systems which recombines the interrogation beams based on theirwavelength or spatial positioning; an optical detector or collection ofoptical detectors to detect the returning portion or portions of theinterrogation beams; and a processing unit for interpreting collectedresults.

According to another aspect, there is provided a smart-detectionphotoacoustic remote sensing (SD-PARS) system for imaging a subsurfacestructure in the sample which leverages wavelength-specific absorptionto encode or suppress spatial information within the sample.

The SD-PARS system comprises an excitation beam or collection of beamsconfigured to generate PARS signals in the sample at an excitationlocation or collection of locations; an interrogation beam or collectionof beams incident on the sample at an interrogation location orcollection of locations; an optical system or collection of systems thatfocuses or directs the excitation beam or collection of beams at a firstfocal point or collection of focal points and the interrogation beam ata second focal point or collection of focal points, the first and secondfocal points or collection of focal points being below the surface ofthe sample; a portion of the interrogation beam or collection of beamsreturning from the sample that is indicative of the generated PARSsignals; and a processing unit for interpreting collected results. Whatsets SD-PARS apart from standard PARS devices is that the detectionwavelength may be purposefully selected such that it suppressesgenerated photoacoustic or PARS signals from a particular region. Forexample, if a desired target is positioned next to a large blood vesselwhich might otherwise overwhelm the signal from the desired target, thedetection wavelength may be selected as to suppress signal from theblood vessel by populating absorption energy levels prior to detection.The suppressed signal may be suppressed about 1% to about 100% relativeto an unsuppressed signal. In other examples, the suppressed signal maybe suppressed about 5% to about 95%, about 10% to about 90%, about 25%to about 75%, or another suitable fraction relative to the unsuppressedsignal.

Embodiments of TA-PARS, TE-PARS, TS-PARS, SE-PARS, SD-PARS and SR-PARSmay comprise several collections of PARS, TE-PARS, SE-PARS, SD-PARS,TA-PARS, TS-PARS, and SR-PARS detection pathways.

PARS pathways may comprise of but are not limited to conventional PARSas described in U.S. Pat. No. 10,117,583, non-interferometric PARS asdescribed in U.S. Pat. No. 10,327,646, camera-based PARS as described inU.S. Pat. No. 10,627,338, coherence-gated PARS as described inInternational Publication No. WO2019/145764, single-source PARS asdescribed in International Patent Application No. PCT/IB2020/051804,filed on Mar. 3, 2020, and the PARS extensions described inInternational Patent Application No. PCT/IB2019/061131, filed on Dec.19, 2019, the entireties of each of which is incorporated by referenceherein.

According to another aspect, there is provided a dual-modalityphotoacoustic remote sensing combined with optical coherence tomography(PARS-OCT) system for imaging a subsurface structure in the sample whichprovides absorption and scattering contrast of the tissue.

The PARS subsystem of the PARS-OCT comprises an excitation beam orcollection of beams configured to generate pressure and temperaturesignals in the sample at an excitation location or collection oflocations; an interrogation beam or collection of beams incident on thesample at an interrogation location or collection of locations; anoptical system or collection of systems that focuses or directs theexcitation beam or collection of beams at a first focal point orcollection of focal points and the interrogation beam at a second focalpoint or collection of focal points, the first and second focal pointsor collection of focal points being below the surface of the sample; aportion of the interrogation beam or collection of interrogation beamsreturning from the sample that is indicative of the generated pressureand temperature signals; an optical detector or collection of opticaldetectors to detect the returning portion or portions of theinterrogation, and a processing unit for interpreting collected results.

The OCT subsystem of the PARS-OCT comprises a light source or collectionof light sources; an interferometer or collection of interferometerseach with a single or multiple of a sample arm and a reference arm wherethe sample arm directs the sample portion of the beam or collection ofbeams to a third focal point and the reference arm directs the referenceportion of the beam or collection of beams into a path of known length;a portion of the light returning from the sample arm that is indicativeof the scattering collected by the sample arm; a portion of the lightreturning from the reference arm that is indicative of the scatteringcollected by the reference arm; an optical detector or collection ofoptical detectors to detect the returning portions from the sample armor arms and reference arm or arms, and the processing unit forinterpreting collected results.

According to another aspect, there is provided an endoscopicphotoacoustic remote sensing combined with optical coherence tomography(EPARS-OCT) device which provides absorption and scattering informationof the sample.

The PARS subsystem of the EPARS-OCT comprises an excitation beam orcollection of beams configured to generate pressure and temperaturesignals in the sample at an excitation location or collection oflocations; an interrogation beam or collection of beams incident on thesample at an interrogation location or collection of locations; a fiberoptic cable or collection of cables having an input end and a detectionend; an optical system or collection of systems that focuses or directsthe excitation beam or collection of beams at a first focal point orcollection of focal points and the interrogation beam at a second focalpoint or collection of focal points, the first and second focal pointsor collection of focal points being below the surface of the sample; aportion of the interrogation beam or collection of interrogation beamsreturning from the sample that is indicative of the generated pressureand temperature signals; an optical detector or collection of opticaldetectors to detect the returning portion or portions of theinterrogation, and a processing unit for interpreting collected results.

The OCT subsystem of the EPARS-OCT comprises a light source orcollection of light sources; an interferometer or collection ofinterferometers each with a single or multiple of a sample arm and areference arm where the sample arm directs the sample portion of thebeam or collection of beams to a third focal point through a fiber opticcable or collection of cables having an input end and a detection endand the reference arm directs the reference portion of the beam orcollection of beams into a path of known length; a portion of the lightreturning from the sample arm that is indicative of the scatteringcollected by the sample arm; a portion of the light returning from thereference arm that is indicative of the scattering collected by thereference arm; an optical detector or collection of optical detectors todetect the returning portions from the sample arm or arms and referencearm or arms, and the processing unit for interpreting collected results.

Embodiments of PARS-OCT may comprise several collections of PARSdetection pathways and OCT detection pathways.

Embodiments of EPARS-OCT may comprise several collections of PARSdetection pathways and OCT detection pathways.

PARS detection pathways may comprise of but are not limited toconventional PARS as described in U.S. Pat. No. 10,117,583, issued Nov.6, 2018, non-interferometric PARS as described in U.S. Pat. No.10,327,646, issued Jun. 25, 2019, camera-based PARS as described in U.S.Pat. No. 10,627,338, issued Apr. 21, 2020, coherence-gated PARS asdescribed in U.S. Publication No. 2020/0359903, published on Nov. 19,2020, single-source PARS as described in WO2020/188386, published Sep.24, 2020, the PARS extensions described in International PatentApplication No. PCT/IB2019/061131, filed on Dec. 19, 2019, TA-PARS,TE-PARS, TS-PARS, SR-PARS, SE-PARS and SD-PARS. All of the patentapplications and patents described in this specification areincorporated herein by reference in their entirety.

OCT detection pathways may comprise of but are not limited to knownimplementations of TD-OCT, SS-OCT, SD-OCT, or other OCT embodiments. Forexample, a TD-OCT system with a broadband light source, scanningreference path delay and a photodetector. For another example, a SS-OCTsystem with tuning narrow band source, stationary reference path delayand a photodetector. For yet another example, a SD-OCT system withbroadband light source, stationary reference path delay and aspectrometer.

Any combinations of the above listed PARS or OCT pathways may beenvisioned such as the specified PARS-OCT. However, any suchcombinations or obvious extensions may also be produced.

Novel PARS signal extraction algorithms may leverage a variety ofabsorption-induced modulation effects including but not limited tomodulation of material reflectivity, scattering, polarization, phaseaccumulation, nonlinear absorption, nonlinear scattering, etc. These maybe used for multiplex acquisitions to unmix constituent chromophoresfrom within a sample by using a variety of excitation, detection beam,and signal enhancement beam properties including but not limited tovariations in wavelength, pulse width, power, energy, coherence length,repetition rate, exposure times, etc. These properties may take on anyvalue appropriate for the task. Common ranges may include: wavelengths(nanometers to microns), pulse widths (attoseconds to milliseconds),powers (attowatts to watts), pulse energies (attojoules to joules),coherence lengths (nanometers to kilometers), and repetition rates(continuous-wave to gigahertz). The excitation beam may generally beimplemented using shorter pulse widths (nanosecond and sub-nanosecond)intended to elicit a PARS signal impulse response, as opposed to thesignal enhancement beam which may be implemented using relatively longerpulse widths (nanosecond and longer) as the signal enhancement beam mayonly need to elicit a thermal perturbation. For example, the pulse widthof the excitation beam may be greater than 1 ns, or less than 1 ns; andthe pulse width of the signal enhancement beam may be higher. In a givensystem architecture the excitation, detection, and signal enhancementwavelengths may be implemented using different wavelengths orpolarization states as to provide a means of optical differentiationbetween the respective pathways.

Other novel PARS signal extraction algorithms may leveragecharacteristic features of collected time-domain behavior to improvesignal fidelity, enhance image contrast and to recover information onthe sample shape, size and dimensions, or for performingmultiplexed/functional imaging. Processing techniques may include butare not limited to lock-in amplification (both software andhardware-based implementations), machine learning methods, broad featureextraction, multidimensional decomposition and frequency content-basedfeature extraction and signal processing methods.

PARS may be used to unmix the composition of targets based on theirabsorption, temperature, polarization, frequency, phase, nonlinearabsorption, constitution, velocity, fluorescence, nonlinear scatteringand scattering content.

It may also be used to unmix the size, shape, feature, and dimensions oftargets based on their absorption, temperature, polarization, frequency,phase, nonlinear absorption, nonlinear scattering and scatteringcontent.

The PARS signals may be used for unmixing targets using their absorptioncontents, scattering contents, fluorescence, polarization contents,frequency contents, phase contents by taking advantage of differentwavelengths, different pulse widths, different coherence lengths,repetition rates, lasers exposure time, laser fluence.

PARS signals may be dominated by generated pressure and analyzed basedon their, amplitude/intensity, frequency content, content related topolarization changes, fluorescence, second harmonic generation, andphase variations to provide information.

PARS signals may be dominated by generated temperature and analyzedbased on their, amplitude/intensity, fluorescence, frequency content,second harmonic generation, content related to polarization changes, andphase variations to provide information.

The PARS system may be configured to capture any optical absorptioninduced variations in the sample. Such variations may include, but arenot limited to, pressure signals, temperature signals, ultrasoundsignals, autofluorescence signals.

A portion of interrogation, signal enhancement, excitation orautofluorescence from the sample may be collected to form images. Thesesignals may be used to unmix the size, shape, feature, dimensions,nature and composition of sample.

In a given architecture, any portion of the light returning from thesample such as the detection, excitation, or thermal enhancement beamsmay be collected. The returning light may be analyzed based onwavelength, phase, polarization, etc. to capture any absorption inducedsignals including, pressure, temperature, and optical emissions. In thisway, the PARS may simultaneously capture for example, scattering,autofluorescence, and polarization contrast attributed to eachdetection, excitation, and thermal enhancement source. Moreover, thePARS laser sources may be specifically chosen to highlight thesedifferent contrast mechanisms.

Other aspects will be apparent from the description and claims below.

BRIEF DESCRIPTION OF THE DRAWINGS

In this patent document, the word “comprising” is used in itsnon-limiting sense to mean that items following the word are included,but items not specifically mentioned are not excluded. A reference to anelement by the indefinite article “a” does not require that there be oneand only one of the elements.

The scope of the following claims should not be limited by the preferredembodiments set forth in the examples above and in the drawings butshould be given the broadest interpretation consistent with thedescription as a whole.

FIG. 1 An overview of the TE-PARS system.

FIG. 2 An overview of the TE-PARS system with PARS Excitation and PARSDetection.

FIG. 3 An overview of a SR-TE-PARS system.

FIG. 4 Shows a possible implementation of TE-PARS being combined withother modalities.

FIG. 5 Shows a signal processing pathway of TE-PARS signals.

FIG. 6 Shows a implementation of multiple PARS excitation lasers.

FIG. 7 Shows a implementation of multiple PARS detection lasers.

FIG. 8 Shows a implementation of multiple PARS signal enhancementlasers.

FIG. 9 Shows a implementation of multiple excitation lasers, multipledetection lasers and multiple signal enhancement lasers.

FIG. 10 Shows an example of a system layout for TE-PARS.

FIG. 11 Shows yet another example of a system layout for TE-PARS.

FIG. 12 Shows yet another example of a system layout for TE-PARS.

FIG. 13 Shows an example of combining TE-PARS with other modalities.

FIG. 14 Shows an example of a TE-PARS imaging method using continuousthermal enhancement.

FIG. 15 Shows an example of a TE-PARS imaging method using pulsedthermal enhancement.

FIG. 16 Shows a diagram of the signal generation in a PARS system.

FIG. 17 Shows a diagram of a TE-PARS signal unmixing method.

FIG. 18 Shows a signal flow for super-resolution imaging.

FIGS. 19 a-k Show different spot arrangements.

FIG. 20 Shows an example of a TE-PARS-based functional imaging method.

FIG. 21 Shows an example of a PARS excitation series and the resultingPARS signal.

FIG. 22 Shows a implementation of the SE-PARS system.

FIG. 23 Shows an overview of the PARS-OCT system.

FIGS. 24 a-b Show implementations of the PARS-OCT system imaging arms.

FIG. 25 Shows implementations of the PARS-OCT system.

FIGS. 26 a-c Shows mechanisms of OCT imaging systems.

FIG. 27 Shows an overview of OCT signal data processing pathway.

FIG. 28 Shows an example OCT image of the human retina (B-scan).

FIG. 29 a-c Show mechanisms of PARS imaging systems.

FIG. 30 Shows an overview of PARS signal data processing pathways.

FIGS. 31 a-b Show example PARS images.

FIG. 32 Shows an example system layout for a PARS-OCT.

FIG. 33 Shows another example system layout for an EPARS-OCT.

FIG. 34 Shows yet another example system layout for a PARS-OCT.

FIG. 35 Shows an example system layout for multimodal PARS-OCT.

FIG. 36 Shows yet another example system layout for a PARS-OCT.

FIG. 37 Shows an overview of a TS-PARS.

FIG. 38 Shows an example combination between a TE-PARS, TS-PARS, SR-PARSand other modalities.

FIG. 39 Shows a PARS-OCT which features a thermal enhancement source.

FIG. 40 Shows a PARS system wherein the optical subsystems are scannedmechanically about the sample.

FIG. 41 Shows an example of the TS-PARS detection process.

FIG. 42 Shows a scattering subtraction processing pathway.

FIGS. 43 a-b Show examples of various constituent optical beamsdemonstrating that some pathways may be implemented in transmissionmode.

FIGS. 44 a-k Show different spot arrangements for a PARS-OCT system.

FIG. 45 Shows another example of a scattering subtraction processingpathway.

FIG. 46 Shows an example of an SD-PARS combined scattering andabsorption contrast visualization.

FIG. 47 Shows an example of the different contrast provided by varyingthe SD-PARS detection source wavelength.

FIG. 48 Shows an example of an autofluorescence sensitive totalabsorption PARS (TA-PARS) architecture.

FIG. 49 Shows an example of a visualization produced by theautofluorescence sensitive total absorption PARS (TA-PARS) architecture.

FIG. 50 shows an example of TA-PARS.

DETAILED DESCRIPTION

Reference will now be made in detail to examples of the presentdisclosure, which are illustrated in the accompanying drawings. Whereverpossible, the same reference numbers will be used throughout thedrawings to refer to the same or like parts. In the discussion thatfollows, relative terms such as “about,” “substantially,”“approximately,” etc. are used to indicate a possible variation in astated numeric value.

Since PARS devices utilized two optical beams which may be in a confocalarrangement, spatial resolution of the imaging technique may be definedas excitation-defined (ED) or interrogation-defined (ID) depending onwhich of the beams provide a tighter focus at the sample. This aspectalso may facilitate imaging deeper targets, beyond the limits ofconventional contact-based OR-PAM devices. This may be accomplished byleveraging a deeply-penetrating (long transport mean-free-path)detection wavelength such as a short-wave infrared (like 1310 nm or 1700nm) which may provide spatial resolution to a depth superior to thatprovided by a given excitation (such as 532 nm) within highly scatteringmedia such as biological tissues. It is worth mentioning, that if morethan two beams are used such that a system consists of more than twofoci at the sample, then obvious extensions of these components would beexpected. For example, if an additional beam which amplifies the signalwithin its focal region is added, it may also contribute towardsdefining the expected resolution of the system.

The PARS systems described herein are fundamentally different from thepreviously described PARS systems. These devices take advantage of novelphysical discoveries to substantially improve on the capabilities ofprevious reports. Included are PARS systems which take advantage ofmaterial temperature dependencies to enhance the absorption contrast (upto around an order of magnitude depending on the material under test)and sensitivity available to PARS acquisitions. Material saturationeffects are leveraged to surpass resolution capabilities provided solelyby diffraction-limited optics. Modern spatial-spectral encodingtechniques are integrated which may improve acquisition efficiency andimaging rate by several orders of magnitude. This comes as a result ofreducing scan dimensionality, allowing for a two-dimensional scan to becompleted on the order of a one-dimensional scan, or a three-dimensionalscan to be completed on the order of a two-dimensional scan, etc. Aswell, we describe novel processing techniques for use in multiplexacquisitions such as separating chromophores using various beamproperties (wavelength, pulse width, power, coherence length, repetitionrates, exposure times, signal frequency content, and optical saturation,scattering, polarization, and phase effects to name a few), andprocessing techniques for extracting additional information fromtime-domain signals.

Possible mechanisms include a pressure-induced refractive-indexmodulation, thermally-induced refractive index modulation, surfaceoscillations, and scatterer position modulation due to confined thermalexpansion.

Refractive index changes due to temperature and pressure rises may inturn affect the scattering of light. In some cases, the detected PARSsignals may be dominated by the generated pressure and or temperature.

Since many of these novel aspects take advantage of fundamentallydifferent physical effects, these additions are highlighted. First,within a highly simplified abstraction, the pressure generated p₀ by asufficiently short optical pulse of fluence ϕ may be defined by thefollowing relationshipp ₀=Γμ_(a)ϕwhere μ_(a) represents the optical absorption within the sample and Γ isknown as the Grüneisen parameter which describes the ratio of materialproperties. However, coincident with this rise in pressure will be arise in temperature brought on by exposure to the multitude of beamswithin the PARS system. This will in turn affect pressure generation bymodifying both the Grüneisen parameter Γ and the optical absorptionμ_(a). This then implies that the efficiency of pressure generation maybe modified through temperature T. In PARS devices the pressure rise p₀is commonly measured as a change in scattering or reflectivity from theexcitation region. This change in optical scattering may result from theelasto-optic effect in which the pressure p₀ modulates the localrefractive index by an amount δn_(eo) following the relationship for agiven detection wavelength λ as

${\delta{n_{eo}\left( {\mu_{a},\phi,\lambda,T} \right)}} = \frac{\epsilon n_{s}^{3}p_{0}}{2\rho v_{s}^{2}}$where ∈ is the elasto-optic coefficient, n_(s) is the unperturbedrefractive index of the sample, ρ is the density, and v_(s) is theacoustic propagation velocity. Likewise, the rise in temperature T willalso generate a modulation in the local refractive index by some amountδn_(T)(λ, T). These effects then compound upon each other and willpartially defined the measured PARS signal S_(PARS) followingS _(PARS)(μ_(a) ,ϕ,λ,T)∝δn _(eo) +δn ₇Therefore, the intensity-modulated PARS signals hold dependence on notonly optical absorption and incident excitation fluence, but also ondetection laser wavelength, fluence and the temperature of the sample.PARS signals may also arise from other effects such as scattererposition modulation and surface oscillations. A similar analog may existfor PARS devices which take advantage of other modulating opticalproperties such as intensity, polarization, frequency, phase,fluorescence, non-linear scattering, non-linear absorption, etc.

As material properties are dependent on ambient temperature, there is acorresponding temperature dependence in the PARS signal. Thesetemperature dependencies may facilitate temperature sensing with PARSsystems.

Temperature dependencies may also facilitate thermally enhancedphotoacoustic remote sensing (TE-PARS) techniques. The TE-PARS systemsmay use a signal enhancement source in addition to the PARS excitationand detection sources. The signal enhancement source may deposit opticalenergy which modifies the local material properties, and therefore theinduced pressure modulations.

At some intensity levels additional saturation effects may also beleveraged. For example, the optical absorption μ_(a) will experiencesaturation at intensity levels I₀ approaching a characteristicsaturation intensity I_(sat) following

$\mu_{a} = \frac{\mu_{a0}}{1 + {I_{0}/I_{sat}}}$where μ_(ao) is the optical absorption of the material prior tosaturation. This produces a nonlinear spatial distribution of signal fora given linear input of excitation intensity. In much the same way thatnonlinear fluorescent effects are leveraged in super resolutionfluorescent microscopes, PARS may likewise leverage this nonlinearsaturation to surpass the λ/2 diffraction resolution limit.

The above mechanisms point to significant sources of scattering positionor scattering cross-section modulation that could be readily measurablewhen the probe beam is focused to sense the confined excitation volume.However, these large local signals are not the only potential source ofPARS signal. Acoustic signals propagating to the surface of the samplecould also result in changes in PARS signal. These acoustic signals cangenerate surface oscillation as well which result in phase modulation ofthe PARS signals.

These generated signals may be intentionally controlled or effected bysecondary physical effects such as vibration, temperature, stress,surface roughness, mechanical bending among others. For example,temperature may be introduced to the sample which may augment thegenerated PARS signals as compared to those which would be generatedwithout having introduced this additional temperature. Another examplemay involve introducing mechanical stress to the sample (such asbending) which may in turn effects the density of the sample and therebyperturbing with the generated PARS signals as compared to those whichwould have been generated without having introduced this mechanicalstress.

Additional contrast agents may be added to the sample to boost thegenerated PARS signals, this includes but not limited to dyes, proteins,specially designed cells, liquids and optical agents or windows. Thetarget may be altered optically to provide optimized results.

The temperature changes directly affect the PARS signal measured by thedetection laser at the excitation location.

The most direct effect, can be described with the following formula:p ₀=Γη_(th)μ_(a) F

Where η_(th) is the percentage of light converted to heat, F is thelocal optical fluence (J/cm2), and the dimensionless Gruneisen parameterΓ is defined as:

$\Gamma = \frac{\beta}{\kappa\rho C_{v}}$

Where β is the thermal coefficient of volume expansion (K−1), κ is theisothermal compressibility (Pa−1), and ρ is the density (kg/m3).

The signal enhancement beam deposits heat modifying the temperaturedependent Gruneisen parameter and other mechanical properties of thematerial at or adjacent to the focal point of the detection beam and/orthe focal point of the signal enhancement beam. This results in a higherp₀ and therefore higher Photoacoustic and PARS signals of as much as anorder of magnitude depending on the material under test, according tothe aforementioned relation. For example, the signal enhancement beammay increase p₀ by at least 2 times, at least 5 times, at least 10times, at least 20 times compared to a value of p₀ in the absence of thesignal enhancement beam. In other examples, the signal enhancement beammay increase p₀ by at least 5 percent, at least 10 percent, at least 25percent, or at least 50 percent.

Another thermal enhancement effect focuses on the detection beam. Theback-reflection of the detection beam depends on the local refractiveindices of the material. The reflective index of materials is alsotemperature dependent. Therefore, we may use the signal enhancementsource to deposit heat modifying the optical properties of the materialby several percent as compared to the unmodified optical properties ofthe material. In turn, this modifies the amplitude of the observed PARSsignals by roughly the square of the optical property modification. Forexample, using a PARS detection sensitive to intensity reflectivityperturbations, a given refractive-index modulation may elicit anincrease in PARS amplitude by the square of that difference, due to therelationship between the back reflected intensity and therefractive-index of the material under tests.

These limited examples highlight some of the more direct signalenhancement effects. However, many additional material properties aretemperature dependent. We could target any of these material propertieswith the signal enhancement source. This may modify the amplitude,frequency content, etc. of the observed PARS signals.

Temperature ranges would remain appropriate to the target. For example,biological samples should only be heated by a few degrees. For example,the temperature increase of the sample at the focal point of the signalenhancement beam may be from 0.1 to 1 Kelvin, 0.1 to 2 Kelvin, 0.1 to 5Kelvin, 0.1 to 10 Kelvin, although other suitable temperature increasesalso are contemplated.

Another aspect which is leveraged by these new disclosures revolvesaround the scattering, polarization, frequency and phase contents ofgenerated PARS signals. Excitation events occur over short time periods,for example less than 100 ns, in which time, the monitored modulationsin detection signal contain a wealth of information. For example, olderPARS techniques which simply monitored intensity back reflection, mayextract the amplitude of these time-domain signals. However, additionalinformation may be extracted from the time-varying aspects of thesignals. For example, some of the scattering, polarization, frequency,and phase content with a PARS signal may be attributed to the size,shape, features, and dimensions of the region which generated thatsignal. This may encode unique/orthogonal additional information withutility towards improving final image fidelity, classifying sampleregions, sizing constituent chromophores and classifying constituentchromophores to name a few. As such techniques may generate independentdatasets for the same interrogated region they may be combined orcompared with each other. For example, frequency information maydescribe the microscopic structures within the sample, this may becombined with conventional PARS which uses scattering modulation tohighlight regions which are both absorbing and of a specific size.

A final aspect for disclosure in this document revolves around thecombination of a PARS device alongside an optical coherence tomography(OCT). OCT is a complementary imaging modality to PARS devices. WhereasPARS techniques provide visualization of optical absorption contrast,OCT imaging devices provide visualization of optical scatteringcontrast. Each approach captures an independent set of information aboutthe sample. For example, PARS may yield high contrast blood vesselinformation with high specificity, and OCT may yield high contrastinformation of the surrounding tissue such as nearby dermal layers.

OCT measurements can be performed using various approaches, either inthe time domain optical coherence tomography (TD-OCT) or in frequencydomain optical coherence tomography (FD-OCT) as described in [US2010/0265511 and US2014/0125952].

In TD-OCT a laser is passed through an interferometer where one arm (thereference arm) is incident on a movable mirror and the other arm (thesample arm) is incident on the sample. Scattering information istypically extracted by scanning a reference path length and recordingthe resulting interferogram pattern on an optical detector such as aphotodiode as a function of that length. The envelope of this patternrepresents a map of the reflectivity within the sample versus depth,generally called an A-scan, with depth resolution given by the coherencelength of the source laser.

FD-OCT is likewise commonly implemented with an interferometer, a samplearm, and a reference arm. It is generally separated into two distinctmethods. The first, spectral-domain optical coherence tomography(SD-OCT) or spectrometer-based OCT, uses a continuous-wave broadbandlight source and achieves spectral discrimination with a dispersivespectrometer in the detector arm. The second, termed swept-sourceoptical coherence tomography (SS-OCT), time-encodes wavenumberreflectivity by rapidly tuning a narrowband source through a broadoptical bandwidth. Both techniques may allow for a dramatic improvementin SNR of up to 15.0-20.0 dB over TD-OCT.

In OCT systems, multiple A-scans are typically acquired while the samplebeam is scanned laterally across the tissue surface, building up atwo-dimensional map of reflectivity versus depth and lateral extenttypically called a B-scan. The lateral resolution of the B-scan isapproximated by the confocal resolving power of the sample arm opticalsystem, which is usually given by the size of the focused optical spotin the tissue.

There has been a great body of work within the OCT field towardsproviding quantitative optical absorption measurement. This is ofparticular interest within the ophthalmic imaging community whichrequires oxygen saturation measurement about the fundus of the eye.There have been several notable works on this topic, however the currentapproach is still incapable of direct optical absorption measurement(unlike PARS modalities). Rather, optical absorption must be inferredthrough the use of a visible probe source which can greatly limit thepenetration depth into the sample. It would be highly beneficial to thebiomedical imaging community to offer an improved optical absorptionmodality.

Given these complementary properties between PARS and OCT, there wouldbe a clear benefit towards augmenting PARS with OCT. Here, noveltechnical details of a dual-modality PARS OCT system are discussed.

FIG. 1 shows a high-level diagram of a TE-PARS system. This consists ofa PARS system (101), an optical combiner (102), a signal enhancementsystem (103) and an imaging head (104). The optical combiner is used tocombine the beams from the PARS system (101) and the Signal EnhancementSystem (103).

FIG. 2 shows a high-level diagram with the PARS Excitation (202), PARSDetection (204) and Optical Combiner (203) delineated. These arecombined with a Signal Enhancement System (201) and Imaging Head (205).

FIG. 3 shows a high-level diagram of a SR-PARS system. This consists ofa Super-Resolution Processing Unit (301), a PARS system (302), anoptical combiner (303), a signal enhancement system (304) and an imaginghead (305).

FIG. 4 shows a high-level embodiment of a TE-PARS system combined withother modalities (405). This consists of a PARS system (401), opticalcombiner (402), signal enhancement system (403), an imaging head (404).These can be combined with a variety of other modalities such as abright-field microscopy, scanning laser ophthalmoscope, ultrasoundimaging, stimulated Raman microscopy, fluorescence microscopy,two-photon and confocal fluorescence microscopy,Coherent-Anti-Raman-Stokes microscopy, Raman microscopy, other PARS,photoacoustic and ultrasound systems, among others.

FIG. 5 shows the signal processing pathway. This consists of an opticaldetector (501), a signal processing unit (502), a digitizer (503), adigital signal processing unit (504) and a signal extraction unit (505).

FIG. 6 shows an embodiment of multiple excitation lasers combined withoptical combiners.

FIG. 7 shows an embodiment of multiple detection lasers combined withoptical combiners.

FIG. 8 shows an embodiment of multiple signal enhancement laserscombined with optical combiners.

FIG. 9 shows an embodiment of multiple excitation lasers, multipledetection lasers and multiple signal enhancement lasers combined withoptical combiners.

FIG. 10 shows one implementation of the TE-PARS. A multi-wavelengthfiber excitation laser (1012) is used to generate PARS signals. Anexcitation beam (1017) passes through a multi-wavelength unit (1040) anda lens system (1042) to adjust its focus on the sample (1018). Theoptical subsystem used to adjust the focus may be constructed bycomponents known to those skilled in the art including but not limitedto beam expanders, adjustable beam expanders, adjustable collimators,adjustable reflective expanders, telescope systems, etc. The acousticsignatures are interrogated using either a short or long-coherencelength probe beam (1016) from a detection laser (1014) that isco-focused and co-aligned with the excitation spots on the sample(1018). The interrogation/probe beam (1016) passes through a lens system(1043), polarizing beam splitter (1044) and quarter wave plate (1056) toguide the reflected light (1020) from the sample (1018) to thephotodiode (1046). However, this architecture is not limited toincluding a polarizing beam splitter (1044) and quarter wave plate(1056). The aforementioned components may be substituted forfiber-based, equivalent components, e.g., a circulator, coupler, WDM,and/or double-clad fiber, that are non-reciprocal elements. Suchelements may receive light from a first path, but then redirect saidlight to a second path. A signal enhancement laser (1061) is used toenhance the PARS signals using a signal enhancement beam (1060). Thesignal enhancement beam (1060) passes through a lens system (1045) toadjust its focus on the sample (1018). The signal enhancement beam(1060) is combined with the interrogation beam (1016) using a beamcombiner (1031). The combined signal enhancement beam (1060) andinterrogation beam (1016) are further combined with the excitation beamusing another beam combiner (1030). The combined beam (1021) is scannedby a scanning unit (1019). This passes through an objective lens (1055)and is focused onto the sample (1018). The reflected beam (1020) returnsalong the same path and is reflected to the signal collection/analysispathway by the polarized beam splitter (1044). The pathway consists of aphotodiode (1046), amplifier (1048), fast data acquisition card (1050)and computer (1052). In some embodiments the signal enhancement beam maynot be directed along the same pathway as the other beams as it does notnecessarily need to be tightly focused on to the sample at theexcitation location. The signal enhancement beam may include any one ormore of the parameters of the interrogation beam of the excitation beam.The signal enhancement beam could be directed from any angle, usingseparate optics. The signal enhancement beam could be focused onunfocused. The signal enhancement beam can be pulsed, continuous and canbe any wavelength depending on the sample.

Beam properties for the signal enhancement beam may be selected in orderto provide the desired enhancement. Wavelength may be selected based onwhat would be appropriate for the desired contrast, within the sametypes of ranges as the excitation. Intensity would likely be comparablylow compared to the other two beams, but again in similar types ofranges.

FIG. 11 shows another embodiment of TE-PARS. This implementation issimilar to the one shown in FIG. 10 but uses a scanning unit (1111) tomove the sample relative to the interrogation spot rather than scanningthe interrogation spot about the sample. Components with similar labelsto those in FIG. 10 serve similar purposes in this architecture.

FIG. 12 shows yet another embodiment of TE-PARS. This implementation issimilar to the one shown in FIG. 10 but adds components to collect andanalyze the signal enhancement beam reflected from the sample (1210).The signal enhancement beam (1260) is passed through a lens system(1245), polarized beam splitter (1259) and quarter-wave plate (1257).The beam is co-focused with the interrogation beam (1216) and excitationbeam (1217) on the sample. The reflected signal enhancement beam (1210)is reflected onto the signal collection pathway which consists of aphotodiode (1258), amplifier (1268), data acquisition card (1269) and acomputer (1270). Note that this particular example highlights anon-interferometric detection, however, signal enhancement detection maytake the form of any previously recited PARS detection pathway includinginterferometric designs. Components with similar labels to those in FIG.10 serve similar purposes in this architecture.

FIG. 13 shows yet another embodiment of a multi-modal TE-PARS system.This implementation is similar to FIG. 10 but adds bright-fielddetection in which a beam combiner (1373) directs light through a tubelens (1371) and onto a camera (1372). Additional modalities such as abright-field microscopy, scanning laser ophthalmoscope, ultrasoundimaging, stimulated Raman microscopy, fluorescence microscopy,two-photon and confocal fluorescence microscopy,Coherent-Anti-Raman-Stokes microscopy, Raman microscopy, other PARS,photoacoustic and ultrasound systems among others, maybe added in thismanner. Components with similar labels to those in FIG. 10 serve similarpurposes in this architecture. Such integrated additional pathways mayneed to operate on narrow wavelength bands which remain open on a uniquepaths towards the sample. This may involve careful selection ofoperating wavelengths between modalities. The potential benefit of suchapproaches is that a single contained device may be able to provide awide complement of different modalities each with their own benefits.For example in FIG. 13 , the addition of a camera allows for atraditional bright-field microscope which may provide different contrastand achieve a different imaging rates as opposed to the cointegratedPARS device.

FIG. 14 shows a comparison between a standard PARS acquisition (left)and a TE-PARS acquisition (right). The TE-PARS acquisition uses theadditional heat (1 milliKelvin to 10 Kelvin, 20 Kelvin, 30 Kelvin, 40Kelvin, 50 Kelvin, 60 Kelvin, 70 Kelvin, 80 Kelvin, 90 Kelvin, 100Kelvin or more) generated by the signal enhancement laser to improvephotoacoustic conversion efficiency resulting in larger modulations upto around an order of magnitude in the back reflected intensity fromthat region. This additional signal may be used to simply enhanceoverall fidelity, or to highlight contrast from a wavelength differentfrom the excitation. In some examples, TE-PARS may enhancesignal-to-noise ratio by at least 5 percent. In some examples, TE-PARSmay achieve a photoacoustic conversion efficiency of up to 1000%. Forexample, here the excitation uses wavelength “a” to capture a baselinePARS signal. Then, another acquisition is taken with the same excitationwavelength but now using a signal enhancement laser emitting wavelength“b”. The difference between these two signals can then be directlyattributed to the absorption at the signal enhancement wavelength.

FIG. 15 shows an example of the signal acquisition process used by aTE-PARS with a pulsed signal enhancement beam. In this example, amid-infrared (MIR) enhancement beam is used between two standard PARSacquisitions. One of these PARS acquisitions directly follows the MIRpulse such that it experiences additional PARS excitation brought on bya temperature rise introduced by the MIR excitation.

FIG. 16 shows an example of the signal acquisition process used by astandard PARS acquisition. Signal generation is solely based onabsorption of the excitation pulse.

FIG. 17 shows an example of the signal acquisition process used by aTE-PARS which is conducting a multiplex acquisition. In conventionalphotoacoustics, multiplex acquisitions are carried out by using multipleexcitation wavelengths. However, a TE-PARS may use a single excitationwavelength alongside different signal enhancement wavelengths. As such,this approach may be used to separate multiple chromophores based ontheir optical absorption at the signal enhancement wavelengths. Thiscould facilitate visualization of independent constituent components(such as chromophores) despite each individual original datasetproviding a super position of these constituents. In some embodiments,the system may unmix different chromophores from each other (as they maybe mixed in a complex tissue) such as hemoglobin, DNA and lipids. Then,they can be given different color maps and can be shown in one image.Furthermore, now the chromophores/targets can be easily distinguished bylooking at the image.

FIG. 18 shows an example of the signal acquisition process used by aSR-PARS. Multiple standard PARS acquisitions are performed at variousknown excitation energies. Due to saturation effects, the observedoutput PARS signals may in turn provide a non-linear relation to theseexcitation energies. Resolutions tighter than the optical diffractionlimit may be achieved with such a system by leveraging nonlinear opticalabsorption contrast effects within the sample such as opticalintensity-induced optical absorption attenuation (sometimes calledphotobleaching), and nonlinear thermal dependencies of materialproperties such as the thermal expansion coefficient. This algorithm mayuse as inputs several scans of a sample of such that non-linear PARSsignal generation may occur across acquisitions allowing for theapplication of a Vandermonde matrix-based process for separating N'thorder power relationships. These non-linear effects can be leveraged byPARS super-resolution processing algorithms to then extract higher orderspatial frequencies resulting in improved resolution which may reachbeyond the optical diffraction limit.

FIG. 19 shows an example of several local spot positioning conditions.Each of the respective beams (1901), (1902), (1903) may represent any ofthe excitation, detection, or signal enhancement beams. FIG. 19 ahighlights an orientation where one of the excitation, detection, andsignal enhancement beams forms a smaller focal spot as compared to theother two beams. Likewise, FIG. 19 b highlights an alternate casewherein two of the excitation, detection, and signal enhancement beamsform smaller focal spots than the third beam. FIG. 19 c exemplifies athird case where in each of the excitation, detection and signalenhancement beams form nearly equivalent focal spots. FIGS. 19 d and 19e , show focal conditions where in the spots of the constituent beams donot perfectly overlap at the focal spot but are displaced in the lateraldirection. In the first case (FIG. 19 d ) a singular beam is displacedwhile the other two remain overlapped, in the second case (FIG. 19 e )all three beams are displaced relative to each other. Similar to FIGS.19 d and 19 e , FIGS. 19 f and 19 g , show focal conditions where thespots of the constituent beams do not overlap at the focal spot but aredisplaced in the axial direction. In the first case (FIG. 19 f ) asingular beam is displaced while the other two remain overlapped, in thesecond case (FIG. 19 g ) all three beams are displaced relative to eachother. These displacements may be any reasonable value depending on therequirements of the imaging session. FIG. 19 h and FIG. 19 i highlightconditions where the central beam axes form an angle between themselvesand between the sample, where this angle may commonly range between 5and 90 degrees with the sample surface. FIG. 19 h highlights the casewhere two of the beams remain co-aligned while the angle of the thirdbeam is modified. FIG. 19 i shows the case where each of the beams holdsan independent angle relative to the others. Finally, FIG. 19 j and FIG.19 k show two different cases for scanning a sample (1910). In FIG. 19 kthe sample is placed directly within the path of the beams,alternatively in FIG. 19 j there is some scattering media or opticalwindow (1911) located in the beam path prior to the sample, an exampleof a common media within the beam path before the sample would be aglass slide or cover slip to contain the sample. This diagram is notmeant to be limiting and has obvious extensions where the systemcomprises more than three beams. These various conditions may becontrolled by adjusting beam alignment into one or more focusing optics.For example, the excitation may be steered to one side relative to theother beams to produce a condition similar to FIG. 19 d . Likewise, theexcitation focus may be moved axially relative to the other two beams toproduce a condition similar to FIG. 19 f It may be desirable topurposely misaligned these foci in some instances such as rapid opticalscanning in which it may be desirable to, for example, have theinterrogation point trail the excitation point to compensate for therapid focal scanning. Angling the beams relative to each other (FIGS. 19h & 19 i) may provide improvements to detection sensitivity and due tothe higher prevalence of a lateral optical scattering as opposed to aback-optical scattering. Another benefit may come from utilizing thetight lateral focus of the one beam to compensate for the comparablypoor axial focus of another beam by having them overlap each other ataround 90 degrees.

FIG. 20 shows another example of signal processing used by a TE-PARS toconduct multiplex acquisitions. Here, thermal effects are used tomeasure the proportion of two constituent chromophores. In one instance(sample 1), the absorption of detection wavelength “c” is higher thanthat of “a” resulting in a lower returned signal at wavelength “c” and ahigher returned signal at wavelength “a”. Likewise, in sample 2 theabsorption of detection wavelength “a” is higher than that of “c”resulting in a higher returned signal at wavelength “c” and a lowerreturned signal at wavelength “a”. These differences in returnedamplitude are primarily attributed to the difference in the opticalabsorption of the target to each of the wavelengths “a” and “c”.Moreover, the proportion of sample 1 and sample 2 may be determinedbased on the proportionality of returned signal at wavelength “a”compared to wavelength “c”. By leveraging the thermal enhancementeffects in this way, it is possible to perform chromophore unmixingutilizing only a single excitation source.

FIG. 21 shows an example of a TE-PARS system which utilizes a customizedexcitation pulse train of varying pulse widths, energies, wavelengths,and pulse timing to induce specific thermal and pressure effects withinthe sample. These pulse trains can be used to shape and design specificcustomized PARS signals. Moreover, such excitation pulse trains may beleveraged to enhance signals from specific chromophores, suppresssignals from specific chromophores, generate signals of a specific shapeand frequency to aid in signal extraction, etc. For example, a secondarypulse may be timed relative to a first pulse such that the relaxationperiod of the first signal is coincident with the peak of the secondsignal as to further augment the overall signal amplitude.

FIG. 22 demonstrates implementations of the SE-PARS detection system.This implementation is utilizing a similar PARS excitation and deliverysystem as those shown in FIG. 10 through FIG. 13 . but adds componentsto encode information relating to the spatial distribution of focalpoints in the focal plane within the spectral domain of the detectionbeam (2270). This may omit the requirement for optical or mechanicalscanning for small field of views, and may facilitate encoding ofspatial information through single fibers where the spatial distributionis encoded within the spectral distribution. The detection beam (2216)is passed through a lens system (2243), polarized beam splitter (2244)and quarter-wave plate (2256). The beam is then passed through adiffractive optic (2232) which laterally spreads the detection beambased on the light wavelength. The broadened beam is then co-focusedwith the excitation beam (2221) on the sample (2218) through thescanning unit (2219) and the objective lens (2255). The reflected signaldetection beam (2220) is reflected onto the signal collection pathwaywhich consists of another diffractive optic (2233) which laterallyspreads the detection beam based on the light wavelength, a lens (2257)to focus the light onto the photodiode array, a photodiode array (2246),amplifier (2248), data acquisition card (2250) and a computer (2252).Components with similar labels to those in FIG. 10 serve similarpurposes in this architecture.

A TE-PARS, TS-PARS, TA-PARS, SE-PARS, SD-PARS or SR-PARS could also beenvisioned which uses a single optical source for all constituent pathsor collection of paths for the PARS excitation, PARS detection, signalenhancement pathways. In any of these modalities one or more of the beampathways may be oriented in transmission mode meaning that collectionoptics are placed on the opposite side of the sample to optics directinglight at the sample.

FIG. 23 shows a high-level overview of a PARS-OCT system. This consistsof a PARS imaging system (2301), an OCT imaging system (2303), anoptical combiner (2302), and an imaging head (2304) which focuses beampaths onto the sample (2305).

FIG. 24 demonstrates implementations of the PARS imaging subsystem(2301) and OCT imaging subsystem (2303). FIG. 24 (a) shows oneimplementation of the PARS subsystem (2301) which consists of one ormore PARS systems (2401) of one or more PARS system configurations (1,2, . . . , N) which may include, but is not limited to: single source,dual source, pulsed detection, etc. The output of which are then coupledthrough optical combiners (2402) which may be implemented with devicessuch as: free space beam combiners, free space dichroic mirrors,fiber-based interferometers, fiber-based couplers, etc. FIG. 24 (b)shows one implementation of the OCT subsystem (2303) which consists ofone or more OCT systems (2404) of one or more OCT system configurations(1, 2, . . . , M) which may include, but is not limited to:spectral-domain OCT (SD-OCT), swept-source OCT (SS-OCT), time-domain OCT(TD-OCT), full-field OCT (FF-OCT), line-field OCT (LF-OCT),polarization-sensitive OCT (PS-OCT), Gabor-domain OCT (GD-OCT), etc. Theoutput of which are then coupled through optical combiners (2403) whichmay be implemented with devices such as: free space beam combiners, freespace dichroic mirrors, fiber-based interferometers, fiber-basedcouplers, etc.

FIG. 25 demonstrates the combination of a PARS subsystem (2501), an OCTsubsystem (2502), as described in FIG. 24 with an additional alternateimaging subsystem (2503). The alternate imaging subsystem may be, but isnot limited to: bright-field microscopy, scanning laser ophthalmoscope,ultrasound imaging, stimulated Raman microscopy, fluorescencemicroscopy, two-photon and confocal fluorescence microscopy,Coherent-Anti-Raman-Stokes microscopy, Raman microscopy, other PARS,photoacoustic and ultrasound systems, etc. These subsystems (2501, 2502,2503) are then combined through optical combiners (2504) which may beimplemented with devices such as: free space beam combiners, free spacedichroic mirrors, fiber-based interferometers, fiber-based couplers,etc.

FIG. 26 describes the mechanism through which OCT signals are generatedand captured. FIG. 26 (a) shows a representative example of a timedomain interferogram for a specific depth. FIG. 26 (b) shows arepresentative example of a Fourier domain interferogram for an OCTsystem. FIG. 26 (c) shows a representative example of an A-scan for anOCT system which attempts to capture scattering contrast in depth.

FIG. 27 describes the signal processing pathway for an example OCTsystem. The OCT optical detector signal (2701) may be captured withdevices including, but not limited to: photodiodes, avalanchephotodiodes, phototubes, photomultipliers, CMOS cameras, CCD cameras(including EM-CCD, intensified-CCDs, back-thinned and cooled CCDs),spectrometers, etc. This signal may then undergo analog signalprocessing (2702), which may include, but is not limited to low-passfiltration, high-pass filtration, amplification, attenuation, etc.Additionally, the signal may also be fed through an alternate path(2703) to highlight different signal characteristics, leveragingalternate analog processing or lack thereof. The processed or/andunprocessed OCT signal then undergoes signal digitization (2704). Thisdigital signal then undergoes digital signal processing (2705), whichmay include, but is not limited to low-pass filtration, high-passfiltration, Hilbert transformation, Fourier transforms, etc. From thisfully processed signal, in some embodiments, key features may beextracted (2706) to produce an OCT image, which may include, but is notlimited to techniques such as: absolute maximum projection, etc.

FIG. 28 depicts a representative OCT image (B-scan) of the human retina.This image was captured with a SS-OCT system, and demonstratessignificant scattering contrast allowing for detailed analysis of retinaphysiology.

FIG. 29 describes the mechanism through which PARS signals are generatedand captured. FIG. 29 (a) shows a representative example of a PARSsignal, excitation laser activation signal, and an interrogation laseractivation signal across time during an example imaging session. In thiscase, the example PARS system is implemented with a CW interrogationlaser, and a pulsed excitation laser. In FIG. 29(a) several time pointsare highlighted to help demonstrate the functionality of this PARSsystem. Here, the interrogation beam is active across all time points.At time point t₁ the excitation laser is inactive, and the measured PARSsignal remains at rest with a constant (DC) offset. At time point t₂ theexcitation laser has just delivered a short pulse and as the sample hasnow been excited a measurable AC waveform can be seen in thephotothermal and photoacoustic signal. At time point t₃ the PARS signalhas now returned to rest as enough time has passed since the excitationpulse. FIG. 29 (b) demonstrates pressure change and refractive indexchange across a boundary layer transition in depth, moving from anon-absorbing medium to an absorbing medium. This represents the samplecharacteristics at time point t₁ and t₃ of FIG. 29 (a). In this examplethere is a constant increase in refractive index once entering theabsorbing medium, and as the sample is at rest there is no pressurechange when entering the absorbing medium. FIG. 29 (c) demonstrates thesample pressure gradient and refractive index across a boundary layertransition in depth, moving from a non-absorbing medium to an absorbingmedium. This represents the sample characteristics at time point t₂ ofFIG. 29 (a). At this timepoint, the sample has just been delivered apulse from the excitation laser which causes a large pressure gradientin depth in the absorbing medium. This large increase in pressure causesa change in the refractive index of the sample, which causes ameasurable change in the returning light intensity of the interrogationlaser. This can be seen demonstrated as an AC waveform in the PARSsignal in FIG. 29 (a).

FIG. 30 describes the signal processing pathway for an example PARSsystem. The PARS optical detector signal (3001) may be captured withdevices including, but not limited to: photodiodes, avalanchephotodiodes, phototubes, photomultipliers, CMOS cameras, CCD cameras(including EM-CCD, intensified-CCDs, back-thinned and cooled CCDs),spectrometers, etc. This signal may then undergo analog signalprocessing (3002), which may include, but is not limited to low-passfiltration, high-pass filtration, amplification, attenuation, etc.Additionally, the signal may also be fed through an alternate path(3003) to highlight different signal characteristics. The processedor/and unprocessed PARS signal then undergoes signal digitization(3004). This digital signal then may undergo digital signal processing(3005), which may include, but is not limited to low-pass filtration,high-pass filtration, Hilbert transformation, Fourier transforms, PARSsignal identification methods, extracting polarization, phase andfrequency content, etc. From this fully processed signal, in someembodiments, key features can be extracted (3006) to produce a PARSimage, which may include, but is not limited to techniques such as:absolute maximum projection, peak frequency, etc.

FIG. 31 depicts two PARS images which implemented different excitationwavelengths to target specific absorbers. FIG. 31 (a) depicts an in vivoPARS image of vasculature in a mouse ear leveraging green light (532 nm)absorption contrast to target hemoglobin. FIG. 31 (b) depicts an ex vivoPARS image of human tissue leveraging ultraviolet light (266 nm)absorption contrast to target DNA.

FIG. 32 highlights one implementation of PARS-OCT. In this example thebeam coming from an interrogation source (3222) passes through anappropriate collimator (3208) and is directed into a polarizedbeam-splitter (3214), quarter-wave-plate (3215), and is directed towardan appropriate dichroic mirror (3216); together with the excitation beam(3228) coming from the pulsed-laser (3230) and appropriate collimator(3212) and toward the sample path. In the OCT subsystem, the beam comingfrom the broadband light source (3225) is directed into afree-space/fiber-coupler beam splitter (3202) with an appropriate splitratio, and is divided into a reference beam (3227) and a sample beam(3226). These systems may require beams which can provide a widespectrum of illumination such as broadband CW sources, or swept sources.Polarization controllers (3204) might be used to maximize interferenceefficiency. The reference beam is collimated by an appropriatecollimator (3203) and goes through a dispersion compensation unit (3205)and using an appropriate lens (3206) focuses on the reference mirror(3207). The sample beam is directed toward an appropriate dichroicmirror (3223) and is combined with the PARS excitation (3228) andinterrogation (3229) beams. All the beams are then directed toward thesample (3220). In this case galvo-scanner mirrors (3217) are used alongwith a pair of telecentric lenses (3218) and objective lens (3219). Thereturning beams are directed toward the dichroic mirror (3223) andseparated into OCT and PARS interrogation beams. The OCT sample beaminterferes with the reference beam, the mixing is detected by theappropriate detector (3201), and the signal is directed toward thecorresponding processor (3224). The PARS interrogation beam is directedtoward an appropriate filter (3209) to filter out the interrogation beamspectral range, and using an appropriate lens (3210) is focused onto thedetector (3211), the signal is post processed in the correspondingprocessor (3221).

FIG. 33 highlights an implementation of EPARS-OCT. This implementationis similar to that from FIG. 32 except the combination of the beams aredelivered to the sample through an endoscope (3331), which comprises butis not limited to a collimator (3332) along with appropriate imagingoptics (3333 & 3334).

FIG. 34 highlights another novel implementation of PARS-OCT where thePARS excitation and OCT light source is shared (3436) where the lightsource may be but is not limited to a nanosecond-pulsed supercontinuumlaser, directed toward the appropriate dichroic mirror (3435) toseparate PARS excitation (3437) and OCT illumination (3438) beams. Ifthe light source (3436) is shared in this way between multiple paths itmay require broadband output which can be filtered down into requiredsubsections (PARS, OCT, etc.) or tunable such that it may field eachrequirement individually. Outside of the broadband nature, it may notrequire any special properties beyond those particular to the individualbeam sources of other system. Such a device layout may providesignificant advantages over architectures which use individual sourcesfor each pathway. Some of these advantages may include overall devicecost, size, ease of maintenance (including items like alignment), etc.For example, system alignment may be made easier since multiple pathwaysmay share the same wavelength reducing undesired chromatic effects. Inthis example the rest of the system embodiment is similar to that fromFIG. 32 .

FIG. 35 highlights yet another implementation of a multi-modal PARS-OCTsystem. This implementation is similar to that from FIG. 32 . However,rather than a single combiner, two beam combiners (3542) are used tocombine the PARS beams, OCT beam and a bright-field microscope beam(3541). Here the bright-field detection is implemented as a tube lens(3540) and a camera (3539). Additional modalities may be added in thismanner such as fluorescence microscope, scanning laser ophthalmoscope,ultrasound imaging, etc.

FIG. 36 highlights another novel implementation of PARS-OCT where thePARS interrogation and OCT light source is shared (3643) where the lightsource may be but is not limited to a continuous wave laser, directedtoward the appropriate beam combiner (3644) to separate PARSinterrogation (3646) and OCT illumination (3645) beams. Such a devicelayout may provide significant advantages over architectures which useindividual sources for each pathway. Some of these advantages mayinclude overall device cost, size, ease of maintenance (including itemslike alignment), etc. For example, system alignment may be made easiersince multiple pathways may share the same wavelength reducing undesiredchromatic effects. In this example the rest of the system embodiment issimilar to that from FIG. 32 .

Likewise, similar combinations of system can be envisioned which use asingle (i.e., exactly and only one) laser source for all three of thePARS excitation, PARS interrogation, and OCT beams.

FIG. 37 highlights a block diagram for a TS-PARS system. This may beimplemented as a conventional PARS system (3702) that is combined (3703)and is directed onto the sample through an imaging head (3704). At leastone notable difference here, involves the addition of a temperaturesensing unit (3701) which can decode the information contained withinthe PARS signal and interpret it to produce an absolute, or relativetemperature measurement of the sample. Individual PARS measurements maybe compared against known signals for a given sample at a giventemperature. Likewise, multiple PARS measurements may be comparedagainst each other for a given sample to produce a relative change intemperature between these multiple measurements. The sensing unit and/orcontroller then converts these relative changes in PARS signals intorelative or absolute temperature measurements.

FIG. 38 highlights a block diagram for a combination between multiple ofthe described systems, in this case between a TS-PARS (3809), SR-PARS(3801), TE-PARS (3808) and a collection of other modalities (3806).These individual systems are combined (3802, 3803, 3804, 3807) anddirected into a singular imaging head (3805) before being directed ontothe sample. Other combinations of systems described herein may besimilarly combined.

FIG. 39 highlights yet another PARS-OCT implementation which features athermal enhancement source. A signal enhancement laser (3947) is used toenhance the PARS signals using a signal enhancement beam (3949). Theenhancement beam coming from the laser passes through an appropriatecollimator (3948), and is combined with the PARS interrogation beam(3929) using an appropriate beam combiner (3950). In this example therest of the system embodiment is similar to that from FIG. 32 .

FIG. 40 highlights anno-OCT implementation wherein the opticalsubsystems are scanned mechanically about the sample. In this example,the imaging head is mounted on a mechanical scan stage (4051) unit whichenables lateral, axial and rotational scans. In this example the rest ofthe system embodiment is similar to that from FIG. 32 .

FIG. 41 provides a high-level description of the temperature-sensingprocessing which may be present in TS-PARS. As sample temperaturechanges (in this case from 25° to) 35° the efficiency of photoacousticpressure generation also changes. In this case, for a constantexcitation energy level (pulses a, b, c, d), the output signal increaseswith rise in temperature. These temperature modulations can then berecorded and fit to expected temperature-dependent models to extractrelative or absolute changes in temperature within the sample.

FIG. 42 shows a high-level overview of the scattering compensationmethod. The PARS signal has a strong dependence on the unperturbedback-scattered light and therefore local scattering efficiency of asample. To decouple the PARS signal amplitude from the local scatteringefficiency the back-scatter amplitude is extracted and removed from thePARS signals using a unique controller for the task, which may be thesame controller as any of the aforementioned or later mentionedcontrollers, or a different controller. The controller subtractscollected scattering contrast from the PARS signal which serves toreduce the background noise from randomly scattered photons andamplifies the signal to noise ratio.

In any of the TE-PARS, TS-PARS, SE-PARS, SD-PARS, TA-PARS, SR-PARS, PARSor OCT-PARS modalities one or more of the beam pathways may be orientedin transmission mode meaning that beam collection optics are placed onthe opposite side of the sample to those optics which are directinglight at the sample. FIG. 43 exemplifies this case, in the example of aTE-PARS system, here the beams (4301), (4302), (4303) each represent anyone of the TE-PARS excitation, detection and signal enhancement beams.In each case, one or more of these beams may be oriented in the same oropposite direction as the other beams. Moreover, these directionalitiesare not intended to be limiting and may be applied to any of theexemplified beam overlap conditions shown in FIG. 21 , or any otherlogical beam positioning. Some potential advantages of this may resultwhen thin samples (<1 mm) are imaged as forward scattering tends to bemore significant as compared to back scattering. As well suchimplementations may help support the use of multiple modalities at thesame time by not requiring all modalities to arrive at the sample viathe same objective lens, greatly improving multiplexing capabilities.

FIG. 44 shows an example of several local spot positioning conditionsfor a PARS-OCT. Each of the respective beams (4401), (4402), (4403) mayrepresent any the excitation, detection, signal enhancement or OCTbeams. FIG. 44 a highlights an orientation where one of the PARS or OCTexcitation, detection, and signal enhancement beams forms a smallerfocal spot as compared to the other two beams. Likewise, FIG. 44 bhighlights an alternate case wherein two of the excitation, detection,and signal enhancement beams form smaller focal spots than the thirdbeam. FIG. 44 b exemplifies a third case where in each of the PARS orOCT excitation, detection and signal enhancement beams form nearlyequivalent focal spots. FIGS. 44 d and 44 e , show focal conditionswhere in the spots of the constituent beams do not perfectly overlap atthe focal spot but are displaced in the lateral direction. In the firstcase (FIG. 44 d ) a singular beam is displaced while the other tworemain overlapped, in the second case (FIG. 44 e ) all three beams aredisplaced relative to each other. Similar to FIGS. 44 d and 44 e , FIGS.44 f and 44 g , show focal conditions where the spots of the constituentbeams do not overlap at the focal spot but are displaced in the axialdirection. In the first case (FIG. 44 f ) a singular beam is displacedwhile the other two remain overlapped, in the second case (FIG. 44 g )all three beams are displaced relative to each other. Thesedisplacements may be any reasonable value depending on the requirementsof the imaging session. FIG. 44 h and FIG. 44 i highlight conditionswhere the central beam axes form an angle between themselves and betweenthe sample, where this angle may commonly range between 5 and 90 degreeswith the sample surface. FIG. 44 h highlights the case where two of thebeams remain co-aligned while the angle of the third beam is modified.FIG. 44 i shows the case where each of the beams holds an independentangle relative to the others. Finally, FIG. 44 j and FIG. 44 k show twodifferent cases for scanning a sample (4410). In FIG. 44 k the sample isplaced directly within the path of the beams, alternatively in FIG. 44 jthere is some scattering media or optical window (4411) located in thebeam path prior to the sample, an example of a common media within thebeam path before the sample would be a glass slide or cover slip tocontain the sample. This diagram is not meant to be limiting and hasobvious extensions where the system comprises more than three beams.

FIG. 45 shows another high-level example of a PARS signal augmentation.As the PARS signal has a strong dependence on the backscattered lightand therefore local scattering properties of a sample, this informationcan be leveraged to reproduce more accurate visualizations of opticalabsorption contrast by removing remaining scattering contrast inherentin the standard PARS acquisition. In some applications, the scatteringcontent from detection, signal enhancement or excitation lasers may becollected separately. These signals may get subtracted or added to thePARS signals and may analyzed separately based on their amplitude,phase, polarization, and frequency content to provide additionalinformation regarding the sample. In the example shown, in order todecouple the PARS signal amplitude from the local scattering efficiencythe local backscattering amplitude is extracted independently of thePARS signal. In this example, both PARS signals appear to have the sameamplitude though there is significantly less backscattering in the “b”instance. To reduce the local scattering effects, the PARS signals arenormalized relative to their measured local scattering amplitude. Asexemplified here this serves to amplify signals with low scatteringamplitude and reduce signals with strong scattering amplitude. Opticalproperties of any of the detection, signal enhancement, or excitationmay be collected including the polarization, frequency, phase content,fluorescence etc. This information may be mixed, subtracted, added orotherwise augmented with the PARS signal to achieve the desired signalmodification result.

In at least some PARS embodiments, perhaps only absorption contrast(from the excitation sources) is measured. In embodiments of SD-PARS,both scattering contrast (detection source only) and absorption contrast(attributed to the excitation and detection sources) are measured. Thiscan be used directly to produce visualizations, i.e. giving a differentcolor to each image (PARS absorption and scattering) and superimposingthe results. Alternatively, the wavelength-specific absorption andscattering can be leveraged to reveal or suppress information within thesample.

In SD-PARS, the detection wavelength may be purposefully selected suchthat it suppresses generated photoacoustic or PARS signals from aparticular region. For example, if a desired target is positioned nextto a large blood vessel which might otherwise overwhelm the signal fromthe desired target, the detection wavelength may be selected as tosuppress signal from the blood vessel by populating absorption energylevels prior to detection. In SD-PARS, the detection wavelength may alsobe selected to highlight scattering contrast attributed to regions ofinterest within the sample. For example, a highly scattered wavelengthcould be selected to highlight morphological structures when imagingtissues.

In SD-PARS, specific signal extraction algorithms may be applied, whichdepend on both the PARS absorption and scattering time-domain behaviors.The SD-PARS interrogation may be selected to highlight sample specificscattering, absorption, fluorescence, polarization contents, frequencycontents, phase contents. This extra information encoded in the SD-PARSinterrogation, may be applied to improve signal fidelity, enhance imagecontrast and to recover information on the sample shape, size anddimensions. SD-PARS interrogation characteristics may be processed in avariety of manners depending on the desired features and application.For example, processing techniques may include machine learning methods,broad feature extraction, multidimensional decomposition and frequencycontent-based feature extraction and signal processing methods. Incontrast to the standard PARS, information attributed to theinterrogation source interactions, and not the PARS excitation event maybe leveraged to enhance processing techniques. This could also beapplied to enhance multiplex imaging, by suppressing signals fromstructures by populating absorption energy levels prior to detection.This may also be applied to highlighting specific scattering featureswithin a sample.

One such implementation of the SD-PARS focuses on unmixing absorptioncontrast attributed to the detection sources. This SD-PARS is processedas follows:

1) PARS absorption signals are collected with the “standard” method ofbandpass filtering raw photodiode output, prior to capturing the signalwith a high frequency digitizer.

2) Characteristic PARS amplitudes are extracted from the digitized timedomain signal. This is done by any one method such as maximumprojection, frequency analysis, feature decomposition, etc.

3) This may conclude one embodiment of PARS processing.

Following this point, the SD-PARS processing may include additionalprocessing compared to the “standard” PARS processing. This processingcan be broken into three general sections:

Section 1: SD-PARS Absorption Signal Processing

The extracted PARS data from 2) above may be analyzed to highlightsignals characteristic of absorption contrast from the detection source.Low amplitude PARS signals attributed to high absorption of thedetection source or low absorption of the excitation source areextracted using a histogram-based processing technique. First, thehistogram undergoes non-linear scaling. Here, a gamma shift is used to‘stretch’ the lower region of the histogram. This shifts the imagecontrast, highlighting low amplitude PARS signals while suppressing highamplitude PARS signals. The results are then windowed based on astatistical measure of the low amplitude PARS signals. This isolates thesignals of interest. The segmented image is then indicative of tissuefeatures with either low excitation absorption or high detectionabsorption.

Section 2: SD-PARS Scattering Signal Processing

To explicitly isolate contrast attributed to absorption of the detectionsource, the optical scattering contrast of the detection is collected.The scattering signals are collected independently from the PARSabsorption signals. Scattering signals are collected by capturing rawunfiltered (unlike traditional PARS signals) photodiode output.Backscattering intensity is determined at each location as a weightedaverage of the photodiodes time domain output.

Section 3: SD-PARS Unmixing

Unmixing is then performed using the detection scattering intensity andcorresponding PARS absorption signals for each location. PARS absorptionsignals are decomposed into a linear weighted sum of the detectionabsorption contrast and the excitation absorption contrast. This followsthe following proportion:PARS_(ABS)∝Det_(Sc)(Det_(abs)+Ext_(abs))

A combined PARS absorption and scattering visualization is produced bygiving a different color range to each of the image (PARS absorption andscattering), where for example low absorption and scattering signals maybe assigned a white color value, and high-valued such signals may beassigned unique colors to emulate a look of a multiple-stained tissuepreparations. In this example, the contrast attributed to a givenlocation would be a combination of the scattering and absorptioncontrast captured at the given location. The differing contrast may becombined by a number of methods such as linear mixing, or non-linearcolor mixing algorithms. The resulting SD-PARS visualization would thenprovide both the absorption and scattering visualizations. In otherexamples, any signal characteristic could be used to define thecoloring. Thresholding based on the amplitude is one possibleimplementation, however, this is not restricted. Coloring could focus onany property such as magnitude, phase, polarization, frequency content,etc. Moreover, the coloring could be performed with a variety ofmethods, such as, a linear or non-linear mixture, or AI basedapproaches.

FIG. 46 shows an example of an SD-PARS combined scattering andabsorption contrast implementation. We may leverage the SD-PARS systemto collect both scattering and absorption signal simultaneously. Here,the wavelength of the SD-PARS detection is selected to specificallytarget optical properties of the sample. This targeted contrast can beused to directly produce augmented visualizations highlighting bothSD-PARS scattering and absorption contrast. When generating combinedvisualizations, the SD-PARS scattering signals may be analyzed based ontheir amplitude, phase, polarization, frequency content etc. to provideinformation. The extracted SD-PARS scattering contrast, may then besubtracted, added or mixed with the PARS absorption signals by a varietyof methods such as linear or non-linear color mixing, or artificialintelligence-based approaches. In the example shown, we captureabsorption contrast of cell nuclei with the PARS FIG. 46 (a).Concurrently, we capture SD-PARS optical scattering contrastindependently of the absorption signals. In this example, we useinfrared light to capture scattering contrast of the tissue containingthe nuclear structures (b). The two visualizations (a) absorption and(b) scattering are then mixed together to form an augmentedrepresentation. Here we now see the tissue structures and theaccompanying nuclear contrast (c). In this case, the two images arecombined using a basic linear mixing technique. However, this could bedone with any number of techniques including non-linear mixing, or AIbased approaches. Moreover, this could target any optical properties ofthe sample including the polarization, frequency, phase content,fluorescence etc. This information may be mixed, subtracted, added orotherwise augmented with the PARS signal to achieve the desired signalmodification result.

FIG. 47 shows an example of the different contrast potential of SD-PARS.Here, the wavelength of the SD-PARS detection is selected to targetdiffering optical properties of the sample. In this example, we capturescattering images in a thin section of preserved human breast tissue. In(a) we use a near infrared 1310 nm SD-PARS detection source, in (b) weuse a visible wavelength 405 nm SD-PARS detection source. Each sourcehighlights unique structures within the tissue sample. This targetedcontrast forms the basis of the SD-PARS mechanism and augmentedvisualizations.

FIG. 48 shows one implementation of the autofluorescence sensitive PARS.A multi-wavelength fiber excitation laser (4812) is used to generatePARS signals. An excitation beam (4817) passes through amulti-wavelength unit (4840) and a lens system (4842) to adjust itsfocus on the sample (4818). The optical subsystem used to adjust thefocus may be constructed by components known to those skilled in the artincluding but not limited to beam expanders, adjustable beam expanders,adjustable collimators, adjustable reflective expanders, telescopesystems, etc. The acoustic signatures are interrogated using either ashort or long-coherence length probe beam (4816) from a detection laser(4814) that is co-focused and co-aligned with the excitation spots onthe sample (4818). The interrogation/probe beam (4816) passes through alens system (4843), polarizing beam splitter (4844) and quarter waveplate (4856) to guide the reflected light (4820) from the sample (4818)to the photodiode (4846). However, this architecture is not limited toincluding a polarizing beam splitter (4844) and quarter wave plate(4856). The aforementioned components may be substituted forfiber-based, equivalent components, e.g., a circulator, coupler, WDM,and/or double-clad fiber, that are non-reciprocal elements. Suchelements may receive light from a first path, but then redirect saidlight to a second path. The interrogation beam (4816) is combined withthe excitation beam using a beam combiner (4830). The combined beam(4821) is scanned by a scanning unit (4819). This passes through anobjective lens (4855) and is focused onto the sample (4818). Thereflected beam (4820) returns along the same path. The reflected beam isfiltered with a beam combiner/splitter (4831) to separate the detectionbeam (4816) from any autofluorescence light returned from the sample.The autofluorescence light (4890) passes through a lens system (4845) toadjust its focus onto the autofluorescence sensitive photodetector(4891). The isolated detection beam (4820) is transmitted through thebeam splitter (4831) towards the signal collection/analysis pathway.Here the returned detection light is redirected by the polarized beamsplitter (4844). The detection pathway consists of a photodiode (4846),amplifier (4848), fast data acquisition card (4850) and computer (4852).The autofluorescence sensitive photodetector may be any such deviceincluding a camera, photodiode, photodiode array etc. Theautofluorescence detection pathway may include morse beam splitters andphotodetectors to further isolate and detect specific wavelengths oflight.

FIG. 49 shows an example of the visualizations which may potentially beprovided by the autofluorescence sensitive, total absorption PARS(TA-PARS). When a sample absorbs light, there is a limited number ofinteractions which may happen. The absorbed energy is converted totemperature and pressure, or to light of a different wavelength. Whilethe temperature and pressure signals are captured by the PARS detectionbeam, the light emissions may be detected by the autofluorescencesensitive PARS. In this way, all absorption of light by the tissues(whether in the form of generated pressure, generated temperature, orfluorescence) may be captured by the PARS system. In this architecture,any portion of the light returning from the sample, excluding thedetection beam, may be collected and analyzed based on wavelength. Byisolating specific wavelengths of light emissions from the sample, wemay visualize specific molecules of interest. For example, we may applythe autofluorescence sensitive PARS to imaging tissues. Here, weselected the PARS excitation to capture absorption contrast of nuclei.In this case, we use a UV excitation to generate pressure andtemperature signals attributed to nuclei in tissues. Concurrently, wecapture the autofluorescence contrast generated by the PARS excitation.In this case, the non-nuclear regions of the tissues, are highlyfluorescent. In this way, we can provide visualizations of nuclear andnon-nuclear structures in tissues simultaneously. Moreover, theresulting visualizations may only require a single excitation wavelengthto capture.

For example, the autofluorescence sensitive PARS could be implementedinto our PARS absorption spectrometer to accurately measure allabsorption of light by a sample. Moreover, we may use theautofluorescence sensitive PARS to measure the proportion of absorbedenergy which is converted to heat and pressure or light respectively.This may enable the most sensitive quantum efficiency measurements todate.

The TA-PARS signal may also be collected on a single detector ashighlighted in FIG. 50 . Given that the salient components of theTA-PARS signal may appear distinct from each other, a single detectormay appropriately characterize these components. For example, theinitial signal level (Scattering) may be indicative of the un-perturbedintensity reflectivity of the detection beam from the sample at theinterrogation location encoding the scatter intensity. Then, followingexcitation by the excitation pulse (at 100 ns in the diagram), PARSexcitation signals related to thermal, temperature, and fluorescence maybe observed as unique overlapping signals (labeled PA and AF in thediagram). If these excited signals are significantly unique from eachother, they may be decomposed from the combined signal to extract thesemagnitudes along with their characteristic lifetimes. This wealth ofinformation may be useful in improving available contrast, providingadditional multiplexing capabilities, and providing characteristicmolecular signatures of constituent chromophores. In addition, such anapproach may provide pragmatic benefits in that only a single detectorand detection path may be required, drastically reducing physicalhardware complexity and cost.

It will be apparent that other examples may be designed with differentfiber-based or free-space components to achieve similar results. Otheralternatives may include various coherence length sources, use ofbalanced photodetectors, interrogation-beam modulation, incorporation ofoptical amplifiers in the return signal path, etc.

During in vivo imaging experiments, no agent or ultrasound couplingmedium are required. However, the target can be prepared with water orany liquid such as oil before non-contact imaging session. As well, insome instances an intermediate window such as a cover slip or glasswindow may be placed between the imaging system and the sample.

All optical sources including but not limited to PARS excitations, PARSdetections, PARS signal enhancements, and OCT sources may be implementedas continuous beams, modulated continuous beams, or short pulsed lasersin which pulse widths may range from attoseconds to milliseconds. Thesemay be set to any wavelength suitable for taking advantage of optical(or other electromagnetic) properties of the sample, such as scatteringand absorption. Wavelengths may also be selected to purposefully enhanceor suppress detection or excitation photons from different absorbers.Wavelengths may range from nanometer to micron scales. Continuous-wavebeam powers may be set to any suitable power range such as fromattowatts to watts. Pulsed sources may use pulse energies appropriatefor the specific sample under test such as within the range fromattojoules to joules. Various coherence lengths may be implemented totake advantage of interferometric effects. These coherence lengths mayrange from nanometers to kilometers. As well, pulsed sources may use anyrepetition rate deemed appropriate for the sample under test such asfrom continuous-wave to the gigahertz regime. The sources may betunable, monochromatic or polychromatic.

The SD-PARS may use a detection wavelength purposefully selected suchthat it suppresses generated PARS signals from a particular region. Forexample, if a desired target is positioned next to a large blood vesselwhich might otherwise overwhelm the signal from the desired target, thedetection wavelength may be selected as to suppress signal from theblood vessel by popuslating absorption energy levels prior to detection.

The TA-PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS, SD-PARS, PARS-OCT orEPARS-OCT subsystems may use any interferometry designs such as a commonpath interferometer (using specially designed interferometer objectivelenses), Michelson interferometer, Fizeau interferometer, Ramseyinterferometer, Fabry-Perot interferometer, Mach-Zehnder interferometer,and optical-quadrature detection. Interferometers may be free-space orfiber-based or some combination. The basic principle is that phase andamplitude oscillations in the probing receiver beam can be detectedusing interferometry and detected at AC, RF or ultrasonic frequenciesusing various detectors.

The TA-PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS or SD-PARS subsystemsmay use and implement a non-interferometry detection design to detectamplitude modulation within the signal. The non-interferometry detectionsystem may be free-space or fiber-based or some combination therein.

The TA-PARS, TE-PARS, TS-PARS, SD-PARS, SR-PARS, SE-PARS, PARS-OCT orEPARS-OCT subsystems may use a variety of optical fibers such asphotonic crystal fibers, image guide fibers, double-clad fibers etc.

The PARS subsystems may be implemented as a conventional photoacousticremote sensing (PARS), non-interferometric photoacoustic remote sensing(NI-PARS), camera-based photoacoustic remote sensing (C-PARS),coherence-gated photoacoustic remote sensing (CG-PARS), single-sourcephotoacoustic remote sensing (SS-PARS), or extensions thereof.

The OCT subsystem may be implemented as spectral-domain opticalcoherence tomography (SD-OCT), swept-source optical coherence tomography(SS-OCT), time-domain optical coherence tomography (TD-OCT), full-fieldoptical coherence tomography (FF-OCT), line-field optical coherencetomography (LF-OCT), polarization-sensitive optical coherence tomography(PS-OCT), Gabor-domain optical coherence tomography (GD-OCT), etc.

In the PARS-OCT and EPARS-OCT, the PARS and OCT subsystems may operateindividually as a single imaging system and acquire images independentlyas a standalone imaging device.

In one example, all beams may be combined and scanned. In this way, PARSexcitations may be sensed in the same area as they are generated andwhere they are the largest. OCT detection may also be performed in thesame location as the PARS to aid in registration. Other arrangements mayalso be used, including keeping one or more of the beams fixed whilescanning the others or vice versa.

Optical scanning may be performed by galvanometer mirrors, MEMS mirrors,polygon scanners, stepper/DC motors, etc.

Mechanical scanning of the sample may be performed by stepper stages, DCmotor stages, linear drive stages, piezo drive stages, piezo stages,etc.

Both the optical scanning and mechanical scanning approaches may beleveraged to produce one-dimensional, two-dimensional, orthree-dimensional scans about the sample. Adaptive optics such as TAGlenses and deformable mirrors may be used to perform axial scanningwithin the sample.

Both optical scanning and mechanical scanning may be combined to form ahybrid scanner. This hybrid scanner may employ one-axis or two-axisoptical scanning to capture large areas or strips in a short amount oftime. The mirrors can potentially be controlled using custom controlhardware to have customized scan patterns to increase scanningefficiency in terms of speed and quality. For example, one optical axiscan be used to scan rapidly and simultaneously one mechanical axis canbe used to move the sample. This may render a ramp-like scan patternwhich can then be interpolation. Another example, using custom controlhardware, would be to step the mechanical stage only when the fast-axishas finished moving yielding a cartesian-like grid which may not needany interpolation.

PARS may provide 3D imaging by optical or mechanical scanning of thebeams or mechanical scanning of the samples or the imaging head or thecombination of mechanical and optical scanning of the beams, optics andthe samples. This may allow rapid structural and function en-face or 3Dimaging.

One or multiple pinholes may be employed to reject out of focus lightwhen optically or mechanically scanning the beams or mechanical scanningof the samples or the imaging head or the combination of mechanical andoptical scanning of the beams, optics and samples. They may improve thesignal to noise ratio of the resulting images.

Beam combiners may be implemented using dichroic mirrors, prisms,beamsplitters, polarizing beamsplitters, WDMs etc.

Beam paths may be focused on to the sample using different opticalpaths. Each of the single or multiple PARS excitation, detection, signalenhancement etc. paths and OCT paths may use an independent focusingelement onto the sample, or all share a single path or any combination.Beam paths may return from the sample using unique optical paths whichare different from those optical paths used to focus on to the sample.These unique optical paths may interact with the sample at normalincidence, or may interact at some angle where the central beam axisforms an angle with the sample surface ranging from 5 degrees to 90degrees.

The beam configurations shown in FIGS. 19 e and 19 f may provide addedspatial rejection of undesired randomly scattered photons, and detectonly photons that have been modulated by the excitation or signalenhancement laser. Since the PARS imaging region is defined by theoverlap of the excitation beam, detection beam and, in the case ofTE-PARS, the thermal enhancement beam and backwards detection/reflectedbeam path, if these paths are all co-aligned, the interrogated region onthe sample may be defined by a lateral radial distribution which iscommonly shorter than the axial distribution. By angling the beamsrelative to each other, shown in FIGS. 19 e and 19 f the overlap may nowbe defined between the combination of two or more radial distributions.This allows for the lateral resolution of one of the beams to improveupon the axial performance provided by another beam. To maximize thiseffect, it may be most advantageous to have the beams evenly distributedin the azimuth and with around 45 degrees each to the sample surface. Insome embodiments the altitude angles may vary amongst the beam paths.

For some applications such as in ophthalmic imaging, the imaging headmay not implement any primary focusing element such as an objective lensto tightly focus the light onto the sample. Instead, the beams may becollimated, or loosely focused (as to create a spot size much largerthan the optical diffraction limit) while being directed at the sample.For example, ophthalmic imaging devices made direct a collimated beaminto the eye allowing the eye's lens to focus the beam on to the retina.

The imaging head may focus the beams into the sample at least to a depthof 50 nm. The imaging head may focus the beams into the sample at mostto a depth of 10 mm. The added depth over previous PARS arises from thenovel use of deeply-penetrating detection wavelengths as describedabove.

Light may be amplified by an optical amplifier prior to interacting witha sample or prior to detection.

Light may be collected by photodiodes, avalanche photodiodes,phototubes, photomultipliers, CMOS cameras, CCD cameras (includingEM-CCD, intensified-CCDs, back-thinned and cooled CCDs), spectrometers,etc.

The detected signals may be amplified by an RF amplifier, lock-inamplifier, trans-impedance amplifier, or other amplifier configuration.

Modalities may be used for A-, B- or C-scan images for in vivo, ex vivoor phantom studies.

The TA-PARS, TE-PARS, TS-PARS, SD-PARS, SR-PARS, SE-PARS, PARS-OCT orEPARS-OCT may take the form of any embodiment common to microscopic andbiological imaging techniques. Some of these may include but are notlimited to devices implemented as a table-top microscope, invertedmicroscope, handheld microscope, surgical microscope, endoscope, orophthalmic devise, etc. These may be constructed based on principlesknown in the art.

The TA-PARS, TE-PARS, TS-PARS, SD-PARS, SR-PARS, SE-PARS, PARS-OCT orEPARS-OCT may be optimized in order to take advantage of a multi-focusdesign for improving the depth-of-focus of 2D and 3D imaging. Thechromatic aberration in the collimating and objective lens pair may beharnessed to refocus light from a fiber into the object so that eachwavelength is focused at a slightly different depth location. Thesechromatic aberrations may be used to encode depth information into therecovered PARS signals which may be later recovered using wavelengthspecific analysis approaches. Using these wavelengths simultaneously mayalso be used to improve the depth of field and signal to noise ratio(SNR) of the PARS images. During imaging, depth scanning by wavelengthtuning may be performed.

PARS methods may provide lateral or axial discrimination on the sampleby spatially encoding detection regions, such as by using severalpinholes, or by the spectral content of a broadband beam.

The TA-PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS, SD-PARS, PARS-OCT orEPARS-OCT systems may be combined with other imaging modalities such asstimulated Raman microscopy, fluorescence microscopy, two-photon andconfocal fluorescence microscopy, Coherent-Anti-Raman-Stokes microscopy,Raman microscopy, other photoacoustic and ultrasound systems, etc. Thiscould permit imaging of the microcirculation, blood oxygenationparameter imaging, and imaging of other molecularly-specific targetssimultaneously, a potentially important task that is difficult toimplement with only fluorescence based microscopy methods. Amulti-wavelength visible laser source may also be implemented togenerate photoacoustic signals for functional or structural imaging.

Polarization analyzers may be used to decompose detected light intorespective polarization states. The light detected in each polarizationstate may provide information about the sample.

Phase analyzers may be used to decompose detected light into phasecomponents. This may provide information about the sample.

The TA-PARS, PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS or SD-PARS systemsmay detect generated signals in the detection beam(s) returning from thesample. These perturbations may include but are not limited to changesin intensity, polarization, frequency, phase, absorption, nonlinearscattering, and nonlinear absorption and could be brought on by avariety of factors such as pressure, thermal effects, etc.

Analog-based signal extraction may be performed along electrical signalpathways. Some examples of such analog devices may include but are notlimited to lock-in amplifiers, peak-detections circuits, etc.

The PARS subsystem may detect temporal information encoded in theback-reflected detection beam. This information may be used todiscriminate chromophores, enhance contrast, improve signal extraction,etc. This temporal information may be extracted using analog and digitalprocessing techniques. These may include but are not limited to the useof lock-in amplifiers, Fourier transforms, wavelet transforms,intelligent algorithm extraction to name a few. In one example, lock indetection may be leveraged to extract PARS signals which are similar toknown expected signals for extraction of particular chromophores such asDNA, cytochromes, red blood cells, etc.

The OCT subsystems may detect generated PARS, thermal and pressuresignals as perturbations to the back-reflected detection beam. Theseperturbations may include changes in intensity, polarization, phase,frequency, absorption, nonlinear scattering, and nonlinear absorption.The OCT subsystem may detect these perturbations by tracking changesover consecutive OCT scans. The OCT subsystems may also detect thevibration or surface oscillations generated by PARS systems.

The OCT and PARS subsystems may be used for detecting sample absorptionproperties through spectroscopic approaches. It can be used fordetecting either PARS induced absorptions, OCT induced absorption orboth.

The imaging head of the system may include close-loop or open-loopadaptive optic components including but not limited to wave-frontsensors, deformable mirrors, TAG lenses, etc. for wave-front andaberration correction. Aberrations may include de-focus, astigmatism,coma, distortion, 3rd-order effects, etc.

The signal enhancement beam may also be used to suppress signals fromundesired chromophores by purposely inducing a saturation effect such asphotobleaching.

Various types of optics may be utilized to leverage their respectiveadvantages. For example, axicons may be used as a primary objective toproduce Bessel beams with a larger depth of focus as compared to thatavailable by standard gaussian beam optics. Such optics may also be usedin other locations within beam paths as deemed appropriate. Reflectiveoptics may also take the place of their respective refractive elements.Such as the use of a reflective objective lens rather than a standardcompound objective lens.

Optical pathways may include nonlinear optical elements for variousrelated purposes such as wavelength generation and wavelength shifting.

Beam foci may overlap at the sample but may also be laterally andaxially offset from each other when appropriate by a small amount.

The TA-PARS, PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS or SD-PARS systemsmay be used as a spectrometer for sample analysis.

Other advantages that are inherent to the structure will be apparent tothose skilled in the art. The embodiments described herein areillustrative and not intended to limit the scope of the claims, whichare to be interpreted in light of the specification as a whole.

Applications

It will be understood that the system described herein may be used invarious ways, such as those purposes described in the prior art, andalso may be used in other ways to take advantage of the aspectsdescribed above. A non-exhaustive list of applications are discussedbelow.

The system may be used for imaging angiogenesis for differentpre-clinical tumor models.

The system may be used for unmixing targets based on their absorption,scattering or frequency contents by taking advantage of differentwavelengths, different pulse widths, different coherence lengths,repetition rates, exposure time, etc.

The system may be used to image with resolution up to and exceeding thediffraction limit.

The system may be used to image anything that absorbs light, includingexogenous and endogenous targets and biomarkers.

The system may have some surgical applications, such as functional andstructural imaging during brain surgery, use for assessment of internalbleeding and cauterization verification, imaging perfusion sufficiencyof organs and organ transplants, imaging angiogenesis around islettransplants, imaging of skin-grafts, imaging of tissue scaffolds andbiomaterials to evaluate vascularization and immune rejection, imagingto aid microsurgery, guidance to avoid cutting critical blood vesselsand nerves.

The system may also have some gastroenterological applications, such asimaging vascular beds and depth of invasion in Barrett's esophagus andcolorectal cancers. Depth of invasion, in at least some embodiments, iskey to prognosis and metabolic potential. This may be used for virtualbiopsy, crohn's diseases, monitoring of IBS, inspection of carotidartery. Gastroenterological applications may be combined or piggy-backedoff of a clinical endoscope and the miniaturized PARS system may bedesigned either as a standalone endoscope or fit within the accessorychannel of a clinical endoscope.

The system may also be used for clinical imaging of micro- andmacro-circulation and pigmented cells, which may find use forapplications such as in (1) the eye, potentially augmenting or replacingfluorescein angiography; (2) imaging dermatological lesions includingmelanoma, basal cell carcinoma, hemangioma, psoriasis, eczema,dermatitis, imaging Mohs surgery, imaging to verify tumor marginresections; (3) peripheral vascular disease; (4) diabetic and pressureulcers; (5) burn imaging; (6) plastic surgery and microsurgery; (7)imaging of circulating tumor cells, especially melanoma cells; (8)imaging lymph node angiogenesis; (9) imaging response to photodynamictherapies including those with vascular ablative mechanisms; (10)imaging response to chemotherapeutics including anti-angiogenic drugs;(11) imaging response to radiotherapy.

The system may also be used for some histopathology imagingapplications, such as frozen pathology, creating H&E-like images fromtissue samples, virtual biopsy, etc. It may be used on various issuescorporations such as formalin-fixed paraffin-embedded tissue blocks,formalin-fixed paraffin-embedded tissue slides, frozen pathologysections, freshly resected specimen, etc. Within these samplesvisualization of macromolecules such as DNA, RNA, cytochromes, lipids,proteins, etc. may be performed.

The system may be useful in estimating oxygen saturation usingmulti-wavelength PARS excitation in applications including: (1)estimating venous oxygen saturation where pulse oximetry cannot be usedincluding estimating cerebrovenous oxygen saturation and central venousoxygen saturation. This could potentially replace catheterizationprocedures which can be risky, especially in small children and infants.

Oxygen flux and oxygen consumption may also be estimated by using PARSimaging to estimate oxygen saturation, and to estimate blood flow invessels flowing into and out of a region of tissue.

The system may be useful in separating salient histological chromophoressuch as cell nuclei and the surrounding cytoplasm by leveraging theirrespective absorption spectra.

The systems may be used for unmixing targets using their absorptioncontents, scattering, phase, polarization or frequency contents bytaking advantage of different wavelengths, different pulse widths,different coherence lengths, repetition rates, fluence, exposure time,etc.

Other examples of applications may include imaging of contrast agents inclinical or pre-clinical applications; identification of sentinel lymphnodes; non- or minimally-invasive identification of tumors in lymphnodes; imaging of genetically-encoded reporters such as tyrosinase,chromoproteins, fluorescent proteins for pre-clinical or clinicalmolecular imaging applications; imaging actively or passively targetedoptically absorbing nanoparticles for molecular imaging; and imaging ofblood clots and potentially staging the age of the clots.

Other examples of applications may include clinical and pre-clinicalophthalmic applications; oxygen saturation measurement and retinalmetabolic rate in diseases such as age related macular degeneration,diabetic retinopathy and glaucoma, limbal vasculature and stem cellsimaging, corneal nerve and neovascularization imaging, evaluatingSchlemm canal changes in glaucoma patients, choroidal neovascularizationimaging, anterior and posterior segments blood flow imaging and bloodflow state.

The system may be used for measurement and estimation of metabolismwithin a biological sample leveraging the capabilities of both PARS andOCT. In this example the OCT may be used to estimate volumetric bloodflow within a region of interest, and the PARS systems may be used tomeasure oxygen saturation within blood vessels of interest. Thecombination of these measurements then provide estimation of metabolismwithin the region.

The system may be used for head and neck cancer types and skin cancertypes, functional brain activities, Inspecting stroke patient'svasculature to help locate clots, monitoring changes in neuronal andbrain function/development as a result of changing gut bacteriacomposition, atherosclerotic plaques, monitoring oxygen sufficiencyfollowing flap reconstruction, profusion sufficiency following plasticor cosmetic surgery and imaging the cosmetic injectables.

The system may be used for topology tracking of surface deformations.For example, the OCT may be used to track the location of the samplesurface. Then corrections may be applied to a tightly focused PARSdevice using mechanisms such as adaptive optics to maintain alignment tothat surface as scanning proceeds.

The system may be implemented in various different form factorsappropriate to these applications such as a tabletop microscope,inverted microscope, handheld microscope, surgical microscope,ophthalmic microscope, endoscope, etc.

Embodiments

A photoacoustic remote sensing and optical coherence tomography systemfor functional, structural, and multiplex visualization of subsurfacestructures in a sample, comprising:

One or more optical sources configured to generate pressure and thermalsignals in the sample at an excitation location;

One or more optical sources configured to generate an interrogation beamor collection of interrogation beams incident on the sample at theexcitation location, a portion of the interrogation beam or collectionof interrogation beams returning from the sample that are indicative ofthe generated pressure and thermal signals;

One or more optical sources configured to generate an interrogation beamor collection of interrogation beams incident on the sample at theexcitation location, a portion of the interrogation beam or collectionof interrogation beams returning from the sample that are indicative ofthe optical scattering;

A detector or collection of detectors configured to detect the returningportion of the interrogation beam or collection of interrogation beams;

An optical system configured to focus the beams into the sample;

A processor configured to calculate an image of the sample based on thedetected portions of the returning portions of the interrogation beamsfrom the sample.

The system including a non-linear optical element configured to generateor modify beam characteristics.

The system wherein one or more of the PARS excitation/interrogation andOCT interrogation use the same optical source.

The system with different embodiments such as tabletop, handheld,surgical microscope, ophthalmic microscope, endoscope.

The system wherein optical sources may be any continuous, pulsed ormodulated source of electromagnetic radiation with wavelengths rangingfrom approximately 50 nm to 100 μm.

The system including a non-linear optical element configured to generateor modify beam characteristics.

The system wherein one or more of the PARSexcitation/interrogation/signal enhancement beams use the same opticalsource.

The system with different embodiments such as tabletop, handheld,surgical microscope, ophthalmic microscope, endoscope.

For some applications the imaging head may not include any focusingelements.

The system wherein the first, second, and third focal points are at adepth below the surface of the sample that is from 50 nm to 10 mm.

The system wherein all of the beams are focused into the sample andcollected from the sample using the same focusing optics.

The system wherein beams are focused into the sample and beams collectedfrom the sample use different focusing optics.

The system wherein focusing optics are normal to the surface.

The system wherein the central axis of the focusing optics form an anglewith the surface normal that is between 0 degrees and 85 degrees.

The system wherein the beam combiner is implemented using free-spaceoptics.

The system wherein the beam complainers implemented using fiber-baseddevices.

The system wherein the imaging head provides optical scanning bygalvanometer mirrors, MEMS mirrors, polygon scanners, stepper/DC motors,etc.

The system wherein a mechanical scanner such as stepper stages, DC motorstages, linear drive stages, piezo drive stages, piezo stages, etc. isused to scan the sample about the imaging head, the imaging head aboutthe sample, or to scan both at the same time.

The system wherein the detector is an interferometer.

The system wherein the detector is a non-interferometric detector

The system wherein the portion of the beams returning from the sampleencode generated pressure and thermal signals as [intensity,polarization, frequency, phase, fluorescence, non-linear scattering,non-linear absorption] variations.

The system wherein the portion of the beams returning from the sampleare amplified by an optical amplifier.

The system wherein adaptive optics elements are used to adjust beamproperties such as aberrations, focus, and to compensate for surfaceroughness.

The system wherein the system is configured to generate the structure ofthe sample through [a glass window, air, water, vacuum, other material]

The system wherein the OCT detection is configured to detect the PARSmodulations within the sample. OCT detection in this case may act as ashort-coherence PARS interferometric detection. This may facilitate theomission of the PARS detection all together or allow for depth-sensitiveoptical absorption recovery from within a sample. This system willdetect PARS initial pressure signals at the origin to provide uniqueinformation about the optical absorption of the sample.

The system wherein the OCT detection is configured to detect thevibration and oscillations generated by PARS signals. This system willdetect the vibrations caused by PARS pressure propagation at the surfaceand subsurface of the samples to provide unique information about theoptical absorption of the sample.

The system wherein the OCT detection is configured to detect thetopology of the sample.

The system wherein the OCT detection is configured to detect the surfaceroughness of the sample.

A dual-modality photoacoustic remote sensing combined with opticalcoherence tomography (PARS-OCT) system for visualizing details in asample, the system comprising: one or more light sources configured togenerate (1) one or more excitation beams configured to generate signalsin the sample at one or more first locations below a surface of thesample; (2) one or more interrogation beams incident on the sample atone or more second locations; (3) a sample beam; and (4) a referencebeam; wherein a portion of the one or more interrogation beams returningfrom the sample is indicative of the generated signals; one or morefirst optical detectors configured to detect a returning portion orportions of the one or more interrogation beams; one or moreinterferometers, each with a sample arm and a reference arm, wherein thesample arm is configured to direct the sample beam from the one or morelight sources to a third location and the reference arm is configured todirect the reference beam from the one or more light sources into apath; wherein a portion of the sample beam returning from the sample armis indicative of scattering collected by the sample arm; wherein aportion of the reference beam returning from the reference arm isindicative of scattering collected by the reference arm; and wherein theinterferometer is configured to detect returning portions from the oneor more sample arms and one or more reference arms.

The PARS-OCT system, wherein the signals generated by the one or moreexcitation beams include ultrasonic signals, thermal signals,photoacoustic signals, and/or pressure signals, and the returningportion or portions of the one or more interrogation beams areindicative of the generated ultrasonic signals, thermal signals,photoacoustic signals, and/or pressure signals.

The PARS-OCT system, further including one or more beam combinersconfigured to combine at least one excitation beam, at least oneinterrogation beam, and/or the sample beam before delivery to thesample.

The PARS-OCT system, wherein the one or more beam combiners areconfigured to direct a returning portion of the at least oneinterrogation beam to the one or more first optical detectors, and alsois configured to direct a returning portion of the sample beam to theinterferometer.

The PARS-OCT system, further including a bright field microscopy lightsource, wherein the one or more beam combiners are configured to combinelight from the bright field microscopy light source with the at leastone excitation beam, at least one interrogation beam, and the samplebeam before delivery to the sample.

The PARS-OCT system, wherein the system is configured to provideabsorption and scattering contrast of the sample.

The PARS-OCT system, further including a scope, wherein the scopeincludes a collimator and imaging optics, wherein the one or moreexcitation beams, the one or more interrogation beams, and/or the samplebeam are passed through the scope before delivery to the sample.

The PARS-OCT system, wherein the one or more light sources includes afirst light source configured to generate the one or more excitationbeams, the sample beam, and the reference beam.

The PARS-OCT system, wherein the one or more light sources includes asecond light source configured to generate the one or more interrogationbeams.

The PARS-OCT system, wherein the one or more light sources includes afirst light source configured to generate the one or more interrogationbeams, the sample beam, and the reference beam.

The PARS-OCT system, further including one or more optical systemsconfigured to focus or direct (1) the one or more excitation beams toone or more first focal points, and (2) the one or more interrogationbeams at one or more second focal points, the one or more first andsecond focal points being below the surface of the sample.

The PARS-OCT system, wherein: the one or more light sources areconfigured to generate one or more signal enhancement beams, incident onthe sample at the one or more first locations; the one or more firstoptical detectors are configured to detect a returning portion of theone or more signal enhancement beams; and the returning portion of theone or more signal enhancement beams returning from the sample isindicative of generated PARS signals.

The PARS-OCT system, wherein the one or more excitation beams includeexactly one wavelength, and the one or more signal enhancement beamsinclude a plurality of wavelengths.

The PARS-OCT system, further including a controller configured todetermine a temperature of the sample based on an intensity of afeedback from the one or more optical detectors.

The PARS-OCT system, further including a processing unit configured toprovide an image with a resolution greater than an optical diffractionlimit by leveraging nonlinear optical absorption contrast effects withinthe sample, wherein the effects include optical intensity-inducedoptical absorption attenuation or photobleaching, and nonlinear thermaldependencies of material properties including the thermal expansioncoefficient, wherein the processing unit is configured use as inputsseveral scans of a sample such that non-linear PARS signal generationoccurs across acquisitions allowing for the application of a Vandermondematrix-based process for separating N'th order power relationships.

The PARS-OCT system, further including one or more optical systemsconfigured to disperse the one or more interrogation beams based onwavelength or spatial positioning of the one or more interrogationbeams; wherein the one or more optical systems are configured torecombine the one or more interrogation beams based on the wavelength orspatial positioning of the one or more interrogation beams.

The PARS-OCT system, further including one or more pinholes or aperturesconfigured to map desired light to the one or more first opticaldetectors when optically or mechanically scanning the beams, or whenmechanically scanning the sample or an image head.

The PARS-OCT system, wherein the interferometer is configured to detectPARS modulations within the sample, or vibration and oscillationsgenerated by the one or more excitation beams such that the OCT may inturn provide optical absorption contrast.

A dual-modality photoacoustic remote sensing combined with opticalcoherence tomography (PARS-OCT) system for visualizing details in asample, the system providing absorption and scattering contrast oftissue, the system comprising: a PARS subsystem including: one or morelight sources configured to generate (1) one or more excitation beamsconfigured to generate ultrasonic signals, thermal signals,photoacoustic signals, and/or pressure signals in the sample at one ormore excitation locations; (2) one or more interrogation beams incidenton the sample at one or more interrogation locations; one or moreoptical systems configured to focus or direct the one or more excitationbeams at the one or more first focal points, and the one or moreinterrogation beams at one or more second focal points, the one or morefirst and second focal points being below the surface of the sample; aportion of one or more interrogation beams returning from the samplethat is indicative of the generated ultrasonic signals, thermal signals,photoacoustic signals, and/or pressure signals; and one or more opticaldetectors configured to detect the returning portion or portions of theone or more interrogation beams; and an OCT subsystem including: one ormore light sources; and one or more interferometers, each with a samplearm and a reference arm, where the sample arm directs a sample portionof the one or more light sources to a third focal point, and thereference arm directs a reference portion of the one or more lightsources into a path of known length; a portion of light returning fromthe sample arm is indicative of the scattering collected by the samplearm; a portion of the light returning from the reference arm isindicative of the scattering collected by the reference arm; wherein theone or more interferometers are configured to detect returning portionsfrom the sample arm and the reference arm wherein (1) the PARS subsystemand the OCT subsystem share at least one light source, or (2) the PARSsubsystem and the OCT subsystem have only separate light sources.

The following applications: imaging histological samples; imaging cellnuclei; imaging proteins; imaging DNA; imaging RNA; imaging lipids;imaging of blood oxygen saturation; imaging of tumor hypoxia; imaging ofwound healing, burn diagnostics, or surgery; imaging ofmicrocirculation; blood oxygenation parameter imaging; estimating bloodflow in vessels flowing into and out of a region of tissue; imaging ofmolecularly-specific targets; imaging angiogenesis for pre-clinicaltumor models; clinical imaging of micro- and macro-circulation andpigmented cells; imaging of the eye; augmenting or replacing fluoresceinangiography; imaging dermatological lesions; imaging melanoma; imagingbasal cell carcinoma; imaging hemangioma; imaging psoriasis; imagingeczema; imaging dermatitis; imaging Mohs surgery; imaging to verifytumor margin resections; imaging peripheral vascular disease; imagingdiabetic and/or pressure ulcers; burn imaging; plastic surgery;microsurgery; imaging of circulating tumor cells; imaging melanomacells; imaging lymph node angiogenesis; imaging response to photodynamictherapies; imaging response to photodynamic therapies having vascularablative mechanisms; imaging response to chemotherapeutics; imagingfrozen pathology samples; imaging paraffin embedded tissues; imagingH&E-like images; imaging oxygen metabolic changes; imaging response toanti-angiogenic drugs; imaging response to radiotherapy; estimatingoxygen saturation using multi-wavelength PARS excitation; estimatingvenous oxygen saturation where pulse oximetry cannot be used; estimatingcerebrovenous oxygen saturation and/or central venous oxygen saturation;estimating oxygen flux and/or oxygen consumption; imaging vascular bedsand depth of invasion in Barrett's esophagus and/or colorectal cancers;functional and structural imaging during brain surgery; assessment ofinternal bleeding and/or cauterization verification; imaging perfusionsufficiency of organs and/or organ transplants; imaging angiogenesisaround islet transplants; imaging of skin-grafts; imaging of tissuescaffolds and/or biomaterials to evaluate vascularization and/or immunerejection; imaging to aid microsurgery; guidance to avoid cutting bloodvessels and/or nerves; imaging of contrast agents in clinical orpre-clinical applications; identification of sentinel lymph nodes; non-or minimally-invasive identification of tumors in lymph nodes; imagingof genetically-encoded reporters, wherein the genetically-encodedreporters include tyrosinase, chromoproteins, and/or fluorescentproteins for pre-clinical or clinical molecular imaging applications;imaging actively or passively targeted optically absorbing nanoparticlesfor molecular imaging; imaging of blood clots; staging an age of bloodclots; remote or non-invasive intratumoural assessment of glucoseconcentration by detection of endogenous glucose absorption peeks;assessment of organoid growth; monitoring of developing embryos;assessment of biofilm composition; assessment of tooth decay; assessmentof non-living structures; evaluating the composition of paintings fornon-invasive confirmation of authenticity; evaluation of archeologicalartifacts; manufacturing quality control; manufacturing qualityassurance; replacing a catheterization procedure; gastroenterologicalapplications; single-excitation pulse imaging over an entire field ofview; imaging of tissue; imaging of cells; imaging of scattered lightfrom object surfaces; imaging of absorption-induced changes of scatteredlight; or non-contact imaging of optical absorption.

The invention claimed is:
 1. A method for visualizing details in asample, the method comprising: generating pressure, temperature, andfluorescence signals in the sample at an excitation location using anexcitation beam, the excitation beam being focused on the sample;interrogating the sample with an interrogation beam directed toward theexcitation location of the sample, the interrogation beam being focusedon the sample; detecting at least a portion of the interrogation beamreturning from the sample, the returned portion of the interrogationbeam being indicative of the generated pressure and temperature signals;detecting fluorescence signals from the excitation location of thesample, simultaneously with detecting the generated pressure andtemperature signals; detecting a combined signal indicative of thereturned portion of the interrogation beam and the fluorescence signals;and decomposing the combined signal to extract (1) a magnitude andcharacteristic lifetime of the returned portion of the interrogationbeam, and (2) a magnitude and characteristic lifetime of thefluorescence signals.
 2. The method of claim 1, further comprisingseparating the returned portion of the interrogation beam from thefluorescence signals using a beam splitter.
 3. The method of claim 1,further comprising directing the fluorescence signals toward a firstdetector, and directing the returned portion of the interrogation beamtoward a second detector.
 4. The method of claim 1, wherein the methodincludes generating the pressure, temperature, and fluorescence signalsin the sample using exactly one excitation beam at exactly onewavelength.
 5. The method of claim 4, further including preparingvisualizations of both nuclear and non-nuclear structures at theexcitation location using the exactly one wavelength for the excitationbeam.
 6. The method of claim 1, further comprising calculating one ormore images of the sample based on both the returned portion of theinterrogation beam and the fluorescence signals.
 7. The method of claim1, further including using exactly one detector for both 1) detectingthe returned portion of the interrogation beam indicative of thegenerated pressure and temperature signals, and 2) the fluorescencesignals.
 8. The method of claim 7, further including decomposing acombined signal to create 1) a first signal representing the returnedportion of the interrogation beam indicative of the generated pressureand temperature signals, and 2) a second signal representing thefluorescence signals.
 9. The method of claim 1, further comprisingco-focusing and co-aligning the interrogation beam and the excitationbeam toward the excitation location.
 10. The method of claim 1, furthercomprising combining the excitation beam and the interrogation beamusing a beam combiner; filtering a reflected beam to separate thereturned portion of the interrogation beam from the fluorescencesignals; directing the fluorescence signals along a first detectionpathway; and directing the returned portion of the interrogation beamalong a second detection pathway.
 11. The method of claim 10, whereinthe first detection pathway includes one or more secondary beamsplitters to further separate one more wavelengths in the fluorescencesignals.
 12. The method of claim 1, further comprising: directing areflected beam to one or more detectors to detect the returned portionof the interrogation beam and the fluorescence signals.
 13. The methodof claim 1, wherein the generating, interrogating, and detecting stepsare performed without an ultrasound coupling medium.
 14. A method forvisualizing details in a sample, the method comprising: generatingpressure, temperature, and fluorescence signals in the sample at anexcitation location using an excitation beam, the excitation beam beingfocused on the sample; interrogating the sample with an interrogationbeam directed toward the excitation location of the sample, theinterrogation beam being focused on the sample; detecting at least aportion of the interrogation beam returning from the sample, thereturned portion of the interrogation beam being indicative of thegenerated pressure and temperature signals; detecting fluorescencesignals from the excitation location of the sample, wherein thegenerating, interrogating, and detecting steps are performed withoutcontacting the sample; and determining (1) a magnitude andcharacteristic lifetime of the returned portion of the interrogationbeam, and (2) a magnitude and characteristic lifetime of thefluorescence signals.
 15. The method of claim 14, wherein thegenerating, interrogating, and detecting steps are performed without anultrasound coupling medium.
 16. The method of claim 14, furthercomprising: combining the excitation beam and the interrogation beamusing a beam combiner; filtering a reflected beam to separate thereturned portion of the interrogation beam from the fluorescencesignals; directing the fluorescence signals along a first detectionpathway; directing the returned portion of the interrogation beam alonga second detection pathway; directing a reflected beam to one or moredetectors to detect the returned portion of the interrogation beam andthe fluorescence signals; and detecting a combined signal indicative ofthe returned portion of the interrogation beam and the fluorescencesignals, wherein determining (1) the magnitude and characteristiclifetime of the returned portion of the interrogation beam, and (2) themagnitude and characteristic lifetime of the fluorescence signalsincludes: decomposing the combined signal to extract (1) the magnitudeand characteristic lifetime of the returned portion of the interrogationbeam, and (2) the magnitude and characteristic lifetime of thefluorescence signals.
 17. A method for visualizing details in a sample,the method comprising: generating pressure, temperature, andfluorescence signals in the sample at an excitation location usingexactly one excitation beam, the excitation beam being focused on thesample; interrogating the sample with an interrogation beam directedtoward the excitation location of the sample, the interrogation beambeing focused on the sample; detecting at least a portion of theinterrogation beam returning from the sample, the returned portion ofthe interrogation beam being indicative of the generated pressure andtemperature signals; detecting fluorescence signals from the excitationlocation of the sample; and determining a magnitude and characteristiclifetime of the fluorescence signals.
 18. The method of claim 17,further comprising determining a visualization, wherein thevisualization includes nuclear structures in the sample based on thedetected heat and pressure signals, and non-nuclear structures in thesample based on the detected fluorescence signals.
 19. The method ofclaim 17, further comprising determining a magnitude and characteristiclifetime of the returned portion of the interrogation beam.
 20. Themethod of claim 19, further comprising: combining the excitation beamand the interrogation beam using a beam combiner; filtering a reflectedbeam to separate the returned portion of the interrogation beam from thefluorescence signals; directing the fluorescence signals along a firstdetection pathway; directing the returned portion of the interrogationbeam along a second detection pathway; directing a reflected beam to oneor more detectors to detect the returned portion of the interrogationbeam and the fluorescence signals; and detecting a combined signalindicative of the returned portion of the interrogation beam and thefluorescence signals, wherein determining (1) the magnitude andcharacteristic lifetime of the returned portion of the interrogationbeam, and (2) the magnitude and characteristic lifetime of thefluorescence signals includes: decomposing the combined signal toextract (1) the magnitude and characteristic lifetime of the returnedportion of the interrogation beam, and (2) the magnitude andcharacteristic lifetime of the fluorescence signals.